The process of reversible phosphorylation: the work of Edmond H. Fischer

Edmond H. Fischer was awarded the 1992 Nobel Prize in Physiology or Medicine for his joint research with Edwin G. Krebs on reversible protein phosphorylation. The two Classics reprinted here relate some of Fischer and Krebs'' early discoveries in their phosphorylase researchPhosphorylase Activity of Skeletal Muscle Extracts (Krebs, E. G., and Fischer, E. H. (1955) J. Biol. Chem. 216, 113–120)Conversion of Phosphorylase b to Phosphorylase a in Muscle Extracts (Fischer, E. H., and Krebs, E. G. (1955) J. Biol. Chem. 216, 121–132)Edmond H. Fischer was born in Shanghai, China in 1920. He was sent to boarding school in Switzerland at age 7, and in 1935, he entered Geneva''s Collège de Calvin. There, he became friends with his classmate Wilfried Haudenschild, and together, they decided that one of them should go into the sciences and the other into medicine so they could cure the world of all ills. Fischer chose science.Open in a separate windowEdmond H. FischerJust before the start of World War II, Fischer completed high school and entered the School of Chemistry at the University of Geneva. He earned two Licences ès Sciences, one in biology, the other in chemistry, and 2 years later, he was awarded a Diploma of “Ingénieur Chimiste.” For his thesis, he worked with Kurt H. Meyer on the purification of amylase from hog and human pancreas, as well as saliva and several strains of bacteria.In 1950, Fischer went to the United States to do a postdoctoral fellowship with Paul Karrer at CalTech. However, when he arrived in Pasadena he received a letter from Journal of Biological Chemistry (JBC) Classic author Hans Neurath (1), chairman of the department of biochemistry at the University of Washington, offering him an assistant professorship in his department. Fischer visited Seattle and accepted the offer, in part because the surrounding mountains, forests, and lakes reminded him of his native Switzerland.Within 6 months of his arrival, Fischer started working on glycogen phosphorylase with Edwin G. Krebs, who was featured in a previous JBC Classic (2). Krebs had trained with JBC Classic authors Carl and Gerty Cori who had discovered that muscle phosphorylase exists in two forms, phosphorylase a, which was easily crystallized and was active without the addition of AMP, and phosphorylase b, a more soluble protein, which was inactive without AMP (3). They believed that AMP served some kind of co-factor function for the enzyme, facilitating its transition between the two forms.However, in Geneva, Fischer had purified potato phosphorylase, which had no AMP requirement. Because it seemed unlikely that muscle phosphorylase but not potato phosphorylase would require AMP as a co-factor, Fischer and Krebs decided to try to elucidate the role of AMP in the phosphorylase reaction. They never discovered what the nucleotide was doing (this problem was solved several years later when Jacques Monod proposed his allosteric model for the regulation of enzymes), but they did discover that muscle phosphorylase was regulated by an enzyme-catalyzed phosphorylation-dephosphorylation reaction.The two JBC Classics reprinted here relate some of Fischer and Krebs'' early discoveries in their phosphorylase research. In the first Classic, the pair performed experiments to determine whether environmental temperature affects the phosphorylase content of skeletal muscle. They were unable to detect any temperature effects, but they did make the surprising discovery that the muscle extracts contained mainly phosphorylase b rather than phosphorylase a. The pair concluded that “If resting muscle contains mainly phosphorylase b… then pronounced activation of the phosphorylase reaction under various conditions is possible.”The second JBC Classic was printed back-to-back with the first. In it, Krebs and Fischer examine the requirements for the phosphorylase conversion and present evidence that the conversion of phosphorylase b to a in cell-free muscle extracts requires a nucleotide containing high energy phosphate and a divalent metal ion. However, they state that “whether this implies that during conversion there is a direct phosphorylation of the enzyme or the formation of an ‘active’ intermediate cannot be stated at this time. It is also possible that the function of ATP is concerned with the synthesis of a prosthetic group.”Similar work was being carried out on liver phosphorylase at approximately the same time by Earl Sutherland. As discussed in a previous JBC Classic (4), Sutherland discovered the second messenger cyclic AMP (cAMP), which he showed promoted the phosphorylation and activation of phosphorylase. The way in which cAMP promoted phosphorylase activation was eventually elucidated when Krebs and Fischer discovered phosphorylase kinase, which was responsible for phosphorylating phosphorylase. Phosphorylase kinase itself existed in a highly activated phosphorylated form and a less active nonphosphorylated form.As a result of the significance of their studies, Krebs and Fischer were awarded the 1992 Nobel Prize in Physiology or Medicine “for their discoveries concerning reversible protein phosphorylation as a biological regulatory mechanism.”In addition to the Nobel Prize, Fischer has received many awards and honors in recognition of his contributions to science. These include the Werner Medal from the Swiss Chemical Society, the Lederle Medical Faculty Award, the Prix Jaubert from the University of Geneva, and jointly with Krebs, the Senior Passano Award and the Steven C. Beering Award from Indiana University. Fischer was elected to the American Academy of Arts and Sciences in 1972 and to the National Academy of Sciences in 1973.¹     

Oligomerization state influences the degradation rate of 3-hydroxy-3-methylglutaryl-CoA reductase.

The steady-state level of the resident endoplasmic reticulum protein, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), is regulated, in part, by accelerated degradation in response to excess sterols or mevalonate. Previous studies of a chimeric protein (HM-Gal) composed of the membrane domain of HMGR fused to Escherichia coli beta-galactosidase, as a replacement of the normal HMGR cytosolic domain, have shown that the regulated degradation of this chimeric protein, HM-Gal, is identical to that of HMGR (Chun, K. T., Bar-Nun, S., and Simoni, R. D. (1990) J. Biol. Chem. 265, 22004-22010; Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). Since the cytosolic domain can be replaced with beta-galactosidase without effect on regulated degradation, it has been assumed that the cytosolic domain was not important to this process and also that the membrane domain of HMGR was both necessary and sufficient for regulated degradation. In contrast to our previous results with HM-Gal, we observed in this study that replacement of the cytosolic domain of HMGR with various heterologous proteins can have an effect on the regulated degradation, and the effect correlates with the oligomeric state of the replacement cytosolic protein. Chimeric proteins that are oligomeric in structure are relatively stable, and those that are monomeric are unstable. To test the hypothesis that the oligomeric state of the cytosolic domain of HMGR influences degradation, we use an "inducible" system for altering the oligomeric state of a protein in vivo. Using a chimeric protein that contains the membrane domain of HMGR fused to three copies of FK506-binding protein 12, we were able to induce oligomerization by addition of a "double-headed" FK506-like "dimerizer" drug (AP1510) and to monitor the degradation rate of both the monomeric form and the drug-induced oligomeric form of the protein. We show that this chimeric protein, HM-3FKBP, is unstable in the monomeric state and is stabilized by AP1510-induced oligomerization. We also examined the degradation rate of HMGR as a function of concentrations within the cell. HMGR is a functional dimer; therefore, its oligomeric state and, we predict, its degradation rate should be concentration-dependent. We observed that it is degraded more rapidly at lower concentrations.     

Looking at DNA through the Electron Microscope: the Work of Jack Griffith

Electron Microscopic Visualization of the RecA Protein-mediated Pairing and Branch Migration Phases of DNA Strand Exchange(Register, J. C., 3rd, Christiansen, G., and Griffith, J. (1987) J. Biol. Chem. 262, 12812–12820)Formation of DNA Loop Replication Fork Generated by Bacteriophage T7 Replication Proteins(Park, K., Debyser, Z., Tabor, S., Richardson, C. C., and Griffith, J. (1998) J. Biol. Chem. 273, 5260–5270)Jack D. Griffith was born in Logan, Utah, in 1942. He attended Occidental College in Los Angeles and received his B.A. in physics in 1964. Griffith then enrolled at the California Institute of Technology where he worked with Journal of Biological Chemistry (JBC) Classic author James Bonner (1) studying chromosome structure with electron microscopy (EM).Open in a separate windowJack GriffithIn the late 1960s, scientists were using a technique called metal shadow casting to visualize molecules via EM. The method involved spraying a layer of metal on the molecule. Because the sample was slightly raised, it got coated with more metal than its supporting film, which allowed the molecule''s outline to be seen with an electron microscope. A variation on the technique in which the sample was coated with denatured protein was used to visualize DNA. This provided a good way to look at the shape of DNA, but the specific proteins bound to the DNA were obscured by the thick coating. For his Ph.D. work, Griffith developed the EM technology needed to directly visualize bare DNA and DNA-protein complexes. His methods involved carefully controlled rotary shadow casting with tungsten and mounting the DNA on very thin carbon films.After graduating in 1969, Griffith did a 1-year postdoctoral fellowship with Benjamin Siegel at Cornell University and a 3-year fellowship with JBC Classic author Arthur Kornberg (2) at Stanford University. Using the methods Griffith developed at Caltech, Griffith, Kornberg, and Joel A. Huberman published a paper showing an EM image of Escherichia coli DNA polymerase I bound to DNA (3). This was not only the first EM image of DNA bound to a known protein, but it also showed that electron microscopy had the potential to provide quantitative information about macromolecular assemblies involving DNA.Griffith stayed at Stanford as a research scientist until 1978 when he became an associate professor at the Lineberger Comprehensive Cancer Center and the Department of Microbiology and Immunology at the University of North Carolina at Chapel Hill. At UNC, he designed a research program in which he used EM and biochemical tools to study DNA. The two JBC Classics reprinted here demonstrate some of those studies.In the first Classic, Griffith and his colleagues investigated the role of the E. coli RecA protein in homologous recombination. RecA catalyzes this process by promoting pairing and strand exchange between homologous DNA molecules. The scientists used EM to follow reactions in three homologous DNA pairs: supertwisted double-stranded (ds) DNA and linear single-stranded (ss) DNA; linear dsDNA and circular ssDNA; and linear dsDNA and colinear ssDNA. They found that all three reactions undergo a three-step pathway. First, the RecA protein-ssDNA filament makes contact with a homologous dsDNA (joining). Second, both DNA partners are at least partially enveloped within the nucleoprotein filament, and if the DNA topology is favorable, exchange of DNA strands then ensues (envelopment/exchange). Finally, upon completion of strand exchange, this complex is resolved and the products are released.In the second Classic, Griffith teamed with JBC Classic author Charles C. Richardson (4) and used EM to examine the architecture of the DNA and DNA-protein intermediates involved in replication reactions employing the T7 replication proteins. This study showed the first direct evidence of the presence of a DNA loop at the replication fork and provided a long sought after proof of the Alberts trombone model of looping of the lagging strand during replication. One of Griffith and Richardson''s co-authors on this paper, Stanley Tabor, is the son of long time JBC editor Herbert Tabor.Griffith remains at the University of North Carolina at Chapel Hill as Kenan Distinguished Professor of Microbiology and Immunology and Biochemistry. He has received many honors and awards for his contributions to science including the Ellison Senior Scholar Award (2001–2005), the ASBMB Herbert A. Sober Award (2002), the Grand Gold Medal of Comenius University, Slovak Republic (2006), and the Glenn Foundation Award (2007). He was also elected to the American Association for the Advancement of Science (2001) and the American Academy of Arts and Sciences (2005) and served on the Journal of Biological Chemistry editorial board from 2002 to 2007.     

The Discovery and Characterization of Molybdopterin: the Work of K. V. Rajagopalan

Hepatic Sulfite Oxidase. A Functional Role for Molybdenum(Cohen, H. J., Fridovich, I., and Rajagopalan, K. V. (1971) J. Biol. Chem. 246, 374–382)Characterization of the Molybdenum Cofactor of Sulfite Oxidase, Xanthine, Oxidase, and Nitrate Reductase. Identification of a Pteridine as a Structural Component(Johnson, J. L., Hainline, B. E., and Rajagopalan, K. V. (1980) J. Biol. Chem. 255, 1783–1786)The Structure of the Molybdenum Cofactor. Characterization of Di(carboxamidomethyl)molybdopterin from Sulfite Oxidase and Xanthine Oxidase(Kramer, S. P., Johnson, J. L., Ribeiro, A. A., Millington, D. S., and Rajagopalan, K. V. (1987) J. Biol. Chem. 262, 16357–16363)K. V. Rajagopalan was born in 1930 in Udupi, a town in South India. He attended Presidency College in Madras (now called Chennai) and graduated with a B.Sc. in Chemistry in 1951. He then enrolled at the University of Madras to do graduate work in the biochemistry department and earned his Ph.D. in 1957. After graduating, Rajagopalan served as an assistant research officer at the Indian Council of Medical Research. Two years later, he started a postdoctoral fellowship in the biochemistry department at Duke University, working with Journal of Biological Chemistry (JBC) Classic author Philip Handler (1).Open in a separate windowK. V. RajagopalanRajagopalan''s initial research project at Duke, carried out in collaboration with JBC Classic author Irwin Fridovich (2), dealt with the competitive inhibition of several enzymes by urea and guanidine. Next, Rajagopalan moved on to studies of aldehyde oxidase and xanthine oxidase and noticed that both enzymes had unusual absorption spectra. Further investigation revealed that they both contained a molybdenum cofactor, something that had not been seen in any other mammalian proteins. Some of Rajagopalan''s electron paramagnetic resonance (EPR) studies on aldehyde oxidase can be found in the JBC Classic on Helmut Beinert (3).These early findings led to an interest in proteins containing molybdenum and formed the basis of Rajagopalan''s future research. In the first JBC Classic reprinted here, Rajagopalan, Fridovich, and Harvey J. Cohen report the discovery of another molybdenum-containing enzyme, sulfite oxidase. The paper documents the presence and function of molybdenum in sulfite oxidase and describes some aspects of its EPR signal. The paper was published along with two other papers (4, 5), which discussed the purification and properties of sulfite oxidase from bovine liver and the nature of its heme prosthetic group.By 1980, several more molecules had been added to the list of molybdenum-containing proteins, but the nature of the cofactor still remained elusive. This was, in part, due to the fact that the activated cofactor was extremely labile in the presence of oxygen and thus very hard to characterize. To circumvent this problem, Rajagopalan developed a method to isolate the oxidized, inactive form of the molybdenum cofactor from sulfite oxidase, xanthine oxidase, and nitrate reductase. His procedure and initial characterization are reported in the second JBC Classic reprinted here. In the paper, Rajagopalan and his colleagues also provided evidence that a pteridine moiety acted as a structural component of the cofactor.Continuing with this work, Rajagopalan was able to isolate two additional stable degradation products (6) and confirm that the molybdenum cofactor consisted of a complex between molybdenum and a unique pterin which he named molybdopterin.In the final JBC Classic reprinted here, Rajagopalan describes the successful isolation of a stable alkylated derivative of molybdopterin, camMPT, from sulfite oxidase and xanthine oxidase by a procedure involving treatment with iodoacetamide under mild denaturing conditions. Structural studies on the product confirmed that molybdopterin is a 6-alkylpterin with a 4-carbon side chain, which has an enedithiol at carbons 1′ and 2′, a hydroxyl at carbon 3′, and a terminal phosphate group.Today, Rajagopalan remains at Duke as James B. Duke Professor of Biochemistry, a position he assumed in 1995. It is interesting to note that, in 1969 when Handler left Duke to become the President of the National Academy of Sciences, he designated Rajagopalan as PI of his National Institutes of Health (NIH) grant. This has subsequently become the longest continuously funded NIH grant, currently in its 62nd year.     

Ligand Binding in Hemoglobin: the Work of Quentin H. Gibson

Oxygen Binding and Subunit Interaction of Hemoglobin in Relation to the Two-state Model(Gibson, Q. H., and Edelstein, S. J. (1987) J. Biol. Chem. 262, 516–519)Ligand Recombination to the α and β Subunits of Human Hemoglobin(Olson, J. S., Rohlfs, R. J., and Gibson, Q. H. (1987) J. Biol. Chem. 262, 12930–12938)Quentin Howieson Gibson was born in Aberdeen, Scotland in 1918. He attended Queen''s University Belfast and received his M.B., Ch.B. degree in 1941, his M.D. in 1944, and his Ph.D. in 1946. After graduating, he became a lecturer in the Department of Physiology at the University of Sheffield and worked his way up to become Professor and Head of the Department of Biochemistry by 1955.Open in a separate windowQuentin H. GibsonIn 1963, Gibson came to the U.S. and joined the faculty of the Graduate School of Medicine at the University of Pennsylvania as a Professor of Physiology. He remained at Penn until 1965 when he became the Greater Philadelphia Professor of Biochemistry, Molecular and Cell Biology at Cornell University. In 1996, Gibson joined the Department of Biochemistry and Cell Biology at Rice University.Gibson is probably best known for his research on the structure and function of hemoglobin. The hemoglobin molecule consists of four globular protein subunits, each of which contains a heme group that can bind to one molecule of oxygen. The binding of oxygen to hemoglobin is cooperative, the first bound oxygen alters the shape of the molecule to increase the binding affinity of the additional subunits. Conversely, hemoglobin''s oxygen binding capacity is decreased in the presence of carbon monoxide because both gases compete for the same binding sites on hemoglobin, carbon monoxide binding preferentially in place of oxygen.Gibson started his hemoglobin studies in graduate school, submitting a thesis titled “Methaemoglobin,” in which he studied the form of hemoglobin where the iron in the heme group is in the Fe³⁺ state rather than the Fe²⁺ state and is thus unable to carry oxygen. He followed this up with research on familial idiopathic methemoglobinemia, a hereditary hematological disease in which hemoglobin is unable to bind to oxygen, causing dyspnea and fatigue after physical exertion. He was able to identify the pathway involved in the reduction of methemoglobin (1), thereby describing the first hereditary disorder involving an enzyme deficiency. As a result, the disease was named “Gibson''s syndrome.” Since then, Gibson has made numerous additional contributions to the study of hemoglobin, some of which are detailed in the two Journal of Biological Chemistry (JBC) Classics reprinted here.In the first Classic, Gibson and Stuart J. Edelstein look at the oxygen binding and subunit interaction in hemoglobin. In 1965, Jacques Monod, Jeffries Wyman, and Jean-Pierre Changeux proposed a model which stated that proteins that exhibit cooperativity can exist in only two conformational states, and the equilibrium between these two states is modified by binding of a ligand, oxygen in the case of hemoglobin (2). This became known as the “concerted” or the “MWC” model, for Monod, Wyman, and Changeux. (More information on Wyman''s research on protein chemistry and allosterism can be found in his JBC Classic (3).)By the mid-1980s, several groups had found evidence that challenged this model as it related to the mechanistic basis of ligand binding by hemoglobin. For example, Frederick C. Mills and Gary K. Ackers reported that the subunit interactions of hemoglobin decreased on binding of the fourth molecule of oxygen to hemoglobin (4). The effect, which they called “quaternary enhancement,” was incompatible with the two-state MWC allosteric model. In the first Classic, Gibson and Edelstein measured the free energy of binding of the fourth oxygen molecule and compared their result of −8.6 kcal/mol with Mills and Acker''s result of −9.3 kcal/mol. Gibson''s smaller value was consistent with other values found in the literature, and it also allowed reasonable representation of the equilibrium curve using the two-state model without invoking quaternary enhancement.In the second JBC Classic, Gibson looks at ligand binding in human hemoglobin. This paper was an extension of an analysis Gibson had done the previous year on ligand rebinding to sperm whale myoglobin (5). In the paper reprinted here, Gibson and his colleagues explored the rebinding of CO, O₂, NO, methyl, ethyl, n-propyl, and n-butyl isocyanide to the isolated α- and β-chains of hemoglobin as well as the intact molecule. From these experiments the researchers were able to determine the differences between the overall rate constants of the two hemoglobin subunits as well as the differences in binding of the various ligands.In recognition of his contributions to science, Gibson has earned many honors including memberships in the Royal Society of London, the National Academy of Sciences, and the American Association for the Advancement of Science. He served as an Associate Editor for the JBC from 1975 to 1994.     

The Role of Hemoglobin's C-terminal Region: the Work of Eraldo Antonini

Studies on Carboxypeptidase Digests of Human Hemoglobin(Antonini, E., Wyman, J., Zito, R., Rossi-Fanelli, A., and Caputo, A. (1961) J. Biol. Chem. 236, PC60–PC63)The Effect of Oxygenation on the Rate of Digestion of Human Hemoglobins by Carboxypeptidases(Zito, R., Antonini, E., and Wyman, J. (1964) J. Biol. Chem. 239, 1804–1808)Eraldo Antonini (1931–1983) received his degree in Medicine and Surgery from the University of Rome in 1955. He then began studying hemoglobin and myoglobin with Alessandro Rossi-Fanelli at the Institute of Biochemistry of the University of Rome and at the Regina Elena Institute for Cancer Research.Open in a separate windowEraldo Antonini (right) in Caprarola in 1971 for Jeffries Wyman''s (left) birthday party, with Robert W. Noble (back).In 1961, Antonini and Rossi-Fanelli published a paper describing the effect on human hemoglobin''s activity when the C-terminal amino acid residues are removed from the molecule''s α and/or β chains. This paper is reprinted here as a Journal of Biological Chemistry (JBC) Classic. In the study, the scientists used carboxypeptidase A to digest the C-terminal tyrosine and histidine on the molecule''s β chain and carboxypeptidase B to remove the C-terminal lysine, tyrosine, and arginine on the molecule''s α chain. The resulting protein appeared intact but had an increased oxygen affinity, lowered cooperativity, and dramatically reduced Bohr effect.This observation inspired Max Perutz, who wrote: “Several years later, my electron density maps showed that these residues form salt bridges with neighboring subunits in deoxyhaemoglobin which get broken on transition to oxyhaemogloblin. Remembering Antonini''s observation, I realized at once that these bridges must represent the additional bonds between the subunits in the T structure predicted by Monod, Wyman and Changeux''s theory of allostery. Antonini had also demonstrated that the release of Bohr protons is colinear with oxygen uptake. When Kilmartin''s and my work proved that most of the Bohr protons originate from the salt bridges, it became clear to me that oxygen uptake is linked to the rupture of these bridges” (1).In the second JBC Classic reprinted here, Antonini follows up on the first paper by doing a reciprocal experiment in which he looks at differences in digestion rates of oxy- and deoxyhemoglobin, reasoning, “If enzymatic modification can affect conformation and changes of conformation resulting from combination with ligand (oxygen), one might expect that the rate of attack on the hemoglobin by the enzymes should depend on the presence or absence of ligand; this would determine conformation, and conformation, in turn, would control the rate.” Again using carboxypeptidases A and B, he showed that the rate of digestion is different for the oxy- and deoxy- forms of the molecule, indicating a differential accessibility of the C-terminal residues to these enzymes.This work was later extended and perfected by John V. Kilmartin on a suggestion by Perutz, who pointed out the crucial role of the C-terminal residues for the molecular mechanism of cooperativity and the Bohr effect. Kilmartin was able to differentiate the role of the C-terminal histidine from that of tyrosine by preparing and characterizing a modified hemoglobin devoid of histidine.Over the next several years, Antonini continued to study hemoglobin, looking at the properties of the α and β chains, the acid-base equilibria of hemoglobin, the Bohr effect and its dependence on temperature, the oxidation-reduction equilibria, ligand-induced conformational changes in hemoglobin, and the kinetics of the reaction of myoblogins and hemoglobins with ligands. This work culminated in the publication of Hemoglobin and Myoglobin in Their Reactions with Ligands in 1971 (2), which was a landmark in the field.In the 1970s, Antonini expanded his scientific interests and started focusing on electron-transfer metalloproteins (such as cytochrome c oxidase) and on proteolytic enzymes. He eventually became Professor of Molecular Biology at the University of Camerino and was later made Professor of Chemistry and Director of the Institute of Chemistry in the Faculty of Medicine and Surgery at the University of Rome. He also received the Feltrinelli Prize from the Accademia Nazionale dei Lincei in 1974.One of Antonini''s coauthors on the two JBC Classics reprinted here is JBC Classic author Jeffries Wyman (3) who came to Rome in 1961 for a week-long visit and ended up remaining for 25 years and working with Antonini. This collaboration produced a series of outstanding papers and conceptual advancements that have had a long lasting influence on protein chemistry.^1,2     

Exploring the world of phospholipids and their interactions with proteins: the work of William Dowhan

The Gene Encoding the Phosphatidylinositol Transfer Protein Is Essential for Cell Growth (Aitken, J. F., van Heusden, G. P., Temkin, M., and Dowhan, W. (1990) J. Biol. Chem. 265, 4711–4717)A Phospholipid Acts as a Chaperone in Assembly of a Membrane Transport Protein (Bogdanov, M., Sun, J., Kaback, H. R., and Dowhan, W. (1996) J. Biol. Chem. 271, 11615–11618)William Dowhan''s curiosity about the connections between phospholipids and proteins associated with them goes back as far as his days as a graduate student with Esmond Snell at the University of California, Berkeley. In these two JBC Classics, his group''s ability to manipulate biochemical and molecular genetics tools to answer fundamental questions about lipid biology shines through. “William Dowhan and his research group have made many contributions to the biochemistry of phospholipid metabolism and membrane biogenesis,” says Robert Simoni at Stanford University.Open in a separate windowBill Dowhan (right) is shown here with the late Chris Raetz (left), who was a longtime collaborator and friend, and his former postdoctoral advisor, the late Gene Kennedy, on the occasion of Kennedy''s 90th birthday in 2009 (photo courtesy of William Dowhan).The first paper, published in 1990, documented the importance of phosphatidylinositol/phosphatidylcholine transfer proteins in vivo. Dowhan''s group, which has been based at the University of Texas Medical School since 1972, used a combination of biochemistry and genetics to clone the protein''s gene. Dowhan had first heard of phospholipid transfer proteins in 1969, when he began his postdoctoral training with Eugene (Gene) Kennedy at Harvard Medical School. At his very first Kennedy lab meeting, the discussion centered around a publication that had just come out (1). The paper described “one of the first observations of proteins in the soluble phase that transferred lipids between bilayers,” recalls Dowhan. “No one could figure out what these proteins really did in vivo, but they knew the proteins had this function” of transferring lipids between membranes.As he moved through his career, Dowhan focused on cloning and characterizing genes and purifying enzymes responsible for phospholipid metabolism in Escherichia coli. Then came a sabbatical in 1983 with Gottfried (Jeff) Schatz at the Biozentrum of the University of Basel in Switzerland, that expanded Dowhan''s research directions into yeast genetics. He says the opportunity to work with Schatz “got me into the possibility of looking for this phosphatidylinositol/phosphatidylcholine transfer protein (PI-TP) in yeast, which I probably would have never done if I hadn''t taken this sabbatical.”Fresh from his sabbatical, Dowhan started tracking down the protein and its gene in vivo. “I submitted a grant at that time with some preliminary data that we had begun to purify to homogeneity the PI-TP from yeast, which had never been done before. Fortunately, we got the grant,” he says.The Dowhan group managed to purify PI-TP from yeast. “The most important part was using basic biochemistry and understanding how to purify proteins before the advent of genetically tagging proteins for affinity chromatography,” explains Dowhan.For the next step in the process of finding the gene for the protein, Dowhan and colleagues had to apply reverse genetics because the yeast genome was not available in the late 1980s. They sequenced the amino terminus of the protein, made the corresponding oligonucleotide probes, tested yeast cDNA libraries with those probes, and pulled out the gene. “We still didn''t know the role PI-TP played in cell function. But now we had the sequence of the gene and the knock-out mutant was not viable,” notes Dowhan. “So we published” the findings.At the same time, Vytas Bankaitis, now at the University of North Carolina, had been working on cloning the SEC14 gene in yeast, which is necessary for vesicular transport. “It turns out we had missed the DNA sequence,” Dowhan says. From Bankaitis'' work, it was obvious that “PI-TP was the product of the SEC14 gene. It all came together in a joint report in Nature. Now we had a function associated with the SEC14 gene, which we didn''t have before,” Dowhan explains (2). “We had a phenotype of a mutant lacking this phospholipid transfer protein, which then stopped vesicular transport.”This initial link between phospholipid metabolism and vesicular transport opened up the field to characterization of the Sec14 protein superfamily in a broad range of biological systems. These proteins contain lipid-binding domains, which sense membrane lipid composition and integrate lipid metabolism and lipid-mediated signaling with an array of cellular processes.The second JBC Classic focused on a different feature of phospholipids: their role in protein folding. Dowhan was fascinated by membrane proteins ever since he was a graduate student and had gone to the Kennedy laboratory as a postdoctoral fellow, intending to purify the membrane component expressed by the lac operon for lactose transport in E. coli. He was unsuccessful because, at that time, the necessary detergents were not available. Once the lactose permease was purified (3), Dowhan noticed in the literature that other researchers mentioned that when the protein was reconstituted in liposomes missing phosphatidylethanolamine, the protein was defective in energy-dependent uphill transport. Dowhan recalls that he wondered, “Was that an artifact of the liposome system or was that also true in vivo?”To get to the bottom of this observation, Dowhan''s group used E. coli to generate null mutants of what were considered to be absolutely essential genes for phospholipid synthesis and cell viability. They created a null mutant of the pssA gene, which encodes the committed step to the synthesis of the major phospholipid, phosphatidylethanolamine. By establishing conditions in which bacterial cells lacking phosphatidylethanolamine remained viable, the investigators were able to identify and characterize different cell phenotypes caused by the missing phospholipid both in vivo and in vitro. In collaboration with Ronald Kaback at UCLA, Dowhan''s group showed that phosphatidylethanolamine was essential for the proper folding of an epitope of lactose permease that was also necessary to support the energy-dependent uphill transport of lactose. “Studies by others have since shown a similar chaperone role for lipids in other bacteria, plants and mammalian cells,” notes Simoni.To obtain their data, the investigators developed a new technique, the Eastern-Western blot. In this method, membrane proteins were delipidated and partially denatured by SDS. The proteins underwent gel electrophoresis and then were transferred to a solid support by Western blotting. A series of individual lipids were then laid over the proteins at a 90° angle so that the investigators could see, after incubating with conformation-specific antibodies, which lipids helped which membrane proteins regain proper conformation.This technique was used to establish that phosphatidylethanolamine was necessary in a late step of folding of lactose permease, but was not necessary to maintain the final folded state. This observation suggested that lipids act as molecular chaperones in helping protein maturation. “This paper set the stage for understanding how lipids affect the topological organization of wild-type proteins in the membrane,” notes Dowhan.Dowhan and his collaborator Mikhail Bogdanov have continued using bacterial mutants in phospholipid metabolism to systematically manipulate the native membrane lipid compositions during the cell cycle. They have analyzed the transmembrane domain orientation of membrane proteins to establish the molecular basis for lipid-dependent organization of lactose permease and other secondary transporters (4).Dowhan says his work has two take-home messages. One is that “Lipids aren''t just glorified biological detergents,” he says. “They have specific roles” in the cell. The other message is in the power of numbers. Dowhan says the more techniques applied to solve a biological mystery, the more likely the mystery will be successfully solved.     

Mycobacterial Glycophosphoinositides: the Work of Clinton E. Ballou

Biosynthesis of Mannophosphoinositides by Mycobacterium phlei. The Family of Dimannophosphoinositides(Brennan, P., and Ballou, C. E. (1967) J. Biol. Chem. 242, 3046–3056)Biosynthesis of Mannophosphoinositides by Mycobacterium phlei. Enzymatic Acylation of the Dimannophosphoinositides(Brennan, P., and Ballou, C. E. (1968) J. Biol. Chem. 243, 2975–2984)Clinton Edward Edgerton Ballou was born in King Hill, Idaho in 1923. After graduating from high school, he enrolled in a premed program at Boise Junior College, but his interests quickly turned to chemistry after he dissected a poorly embalmed cat in a comparative anatomy course. During his sophomore year, Ballou transferred to Oregon State College in Corvallis where he became involved in two research projects: the first during his sophomore year synthesizing new antimalarial drugs with Bert Christensen, and the second during his senior year studying the guinea pig “antistiffness factor” with Willem van Wagtendonk.Open in a separate windowClinton E. BallouThe military draft was in effect as Ballou entered his last year of college so he decided to join the U. S. Navy after graduating in 1944. He was discharged 2 years later and decided to apply for graduate study in biochemistry with Karl Paul Link at the University of Wisconsin-Madison. As detailed in a previous Journal of Biological Chemistry (JBC) Classic (1), Link''s research centered on blood anticoagulants, and when Ballou arrived in his laboratory in 1946, the primary focus was the structure-function relationship of coumarin anticoagulants. Ballou was immediately intrigued when he learned of a failed attempt to synthesize the glucoside of dicumarol because the acetylated intermediate was degraded in the alkali conditions used for deacetylation. Because glycosides are acetals, which are typically acid-labile and alkali-stable, Ballou decided to study a variety of synthetic compounds to try to understand the structural basis for alkali sensitivity. This research formed the core of his doctoral dissertation, and his exposure to carbohydrate chemistry influenced the direction of his career.After earning his Ph.D. in 1950, Ballou did a year-long postdoctoral fellowship with E. L. Hirst in the Department of Chemistry at the University of Edinburgh. There he studied the structure of maple sapwood starch. At the end of the year, Ballou returned to the U. S. to work with Hermann O. L. Fischer (the son of Nobel laureate Hermann Emil Fischer) at the University of California, Berkeley. Ballou explained his choice: “I was attracted to Fischer in part because of his research on phosphorylated sugars but also because during graduate school I had drawn heavily on the published works of his father, Emil Fischer. I guess the idea of being associated with the son of Emil Fischer just seemed ‘real cool’ to me” (2).The 1950s was a time of active research on biosynthetic pathways involving short chain phosphorylated sugars, and collaborating with Fischer and Donald MacDonald, Ballou undertook the syntheses of several such metabolic intermediates, including d-glyceric acid 2-phosphate, d-glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, hydroxypyruvic acid 3-phosphate, and d-erythrose 4-phosphate. He also became interested in inositol chemistry as a result of studies on the cyclitols in sugar pine heartwood.In 1955, Ballou was appointed to the biochemistry faculty at Berkeley and went about setting up an independent research program. He decided to work on inositol-containing phospholipids and was able to synthesize and characterize d-myoinositol 1-phosphate. He also spent several years isolating and characterizing myoinositol polyphosphates from beef brain phosphoinositide. This culminated in his discovery of d-myoinositol l,4,5-trisphosphate or IP₃. More information on Ballou''s studies of these phosphoinositides can be found in his JBC Reflections (2).Ballou became eligible for sabbatical leave in 1961 and decided to spend a year at the National Center for Scientific Research (CNRS) in France studying the glycophosphoinositides of mycobacteria with Edgar Lederer. There he collaborated with Erna Vilkas on experiments to establish the linkages of both the phosphatidyl and the mannosyl groups to the myoinositol ring (3). Upon his return to Berkeley, Ballou and Yuan Chuan Lee determined the structures of the family of mannosyl phosphoinositides in Mycobacterium smegmatis (4–6). Ballou and his postdoctoral fellow Patrick Brennan then began to look at the biosynthesis of the intact dimannophosphoinositides on Mycobacterium phlei, which is the subject of the two JBC Classics reprinted here.In the first Classic, Ballou and Brennan used subcellular fractions of M. phlei to catalyze the biosynthesis of several mannosyl derivatives of phosphatidylmyoinositol and reported that the major products are a family of three dimannophosphoinositides (A, B, and C) that differ in the number of fatty acyl groups they contain (four, three, and two, respectively). On the basis of their results, they proposed that guanosine diphosphate mannose acts as the sugar donor in the conversion of phosphatidylmyoinositol to phosphatidylmyoinositol dimannoside (dimannophosphoinositide C), which is then acylated in a two-step process to first yield dimannophosphoinositide B and then dimannophosphoinositide A.In the second JBC Classic, Ballou and Brennan provide further evidence for their proposed biosynthesis scheme by using an enzyme from M. phlei to specifically incorporate labeled fatty acids into the dimannophosphoinositides. They showed that label from [¹⁴C]palmityl-CoA is incorporated into dimannophosphoinositide C to yield dimannophosphoinositide B. After a short incubation period, this molecule is converted to dimannophosphoinositide A, but with longer incubation periods the product is deacylated to isomeric forms of dimannophosphoinositides B and C.Brennan continued to work on these glycophosphoinositides after completing his postdoctoral fellowship with Ballou and eventually showed that the lipoglycans (lipomannan (LM) and lipoarabinomannan (LAM)) were multiglycosylated extensions of Ballou''s phosphatidylinositol mannosides and are very important in the pathogenesis of tuberculosis and leprosy. More recent research has defined the biochemistry and genetics of synthesis of these molecules. Brennan is currently University Distinguished Professor in the Department of Microbiology, Immunology, and Pathology at Colorado State University. He served on the editorial board of the Journal of Biological Chemistry for several years and was also named Colorado State University Researcher of the Year in 1992.In 1991, Ballou became Professor Emeritus of Biochemistry at the University of California, Berkeley, although he continued research and teaching for a few years. In recognition of his contributions to science, Ballou has received many awards and honors including election to the National Academy of Sciences (1975), the American Chemical Society''s Claude Hudson Award in Carbohydrate Chemistry (1981), the Welch Foundation Lectureship (1972), the University of Notre Dame Reilly Lectureship (1976), the Duke University Belfort Lectureship (1977), a National Science Foundation Senior Fellowship (1961), and a University of California Berkeley Citation (1992). Ballou also served as an editorial board member for the Journal of Biological Chemistry.     

The molecular genetics of bacteriophage: the work of Norton Zinder

In 1966, Norton Zinder and Joshua Lederberg discovered that Salmonella could exchange genes via bacteriophages. They named this phenomenon “genetic transduction.” This discovery set Zinder on a lifelong journey researching bacteriophage. In the two Journal of Biological Chemistry (JBC) Classic papers reprinted here, Zinder and Nina Fedoroff present their findings on the phage f2 replicase.Properties of the Phage f2 Replicase. I. Optimal Conditions for Replicase Activity and Analysis of the Polynucleotide Product Synthesized in Vitro (Fedoroff, N. V., and Zinder, N. D. (1972) J. Biol. Chem. 247, 4577–4585)Properties of the Phage f2 Replicase. II. Comparative Studies on the Ribonucleic Acid-dependent and Poly(C)-dependent Activities of the Replicase (Fedoroff, N. V., and Zinder, N. D. (1972) J. Biol. Chem. 247, 4586–4592)Norton David Zinder was born in New York City in 1928. He attended the prestigious Bronx High School of Science and went on to Columbia University where he received his B.A. in biology in 1947. Zinder then joined the graduate program at the University of Wisconsin-Madison, studying under geneticist Joshua Lederberg.Open in a separate windowNorton ZinderLederberg recently had found that Escherichia coli could mate and exchange genes (conjugation), a discovery for which he would be awarded the 1958 Nobel Prize in Physiology or Medicine. Zinder''s assignment was to continue Lederberg''s investigations using Salmonella. To do this, he needed to obtain large numbers of mutant bacteria. Rather than using the traditional method of exposing the Salmonella to mutagens and testing the survivors, Zinder decided to use a nutritionally deficient medium and penicillin (negative selection) to select for mutants (1). However, when he began investigating conjugation in Salmonella, most of his attempts at crossing the mutants failed. Fortunately, one mutant strain produced some prototrophs; but puzzlingly, Zinder''s markers did not segregate. Further experiments showed that the mutants were exchanging genes via bacteriophages (2). Lederberg and Zinder named this new phenomenon “genetic transduction.”Zinder received his M.S. in genetics in 1949 and completed his Ph.D. in medical microbiology in 1952. He then accepted a position as assistant professor at Rockefeller University (then known as Rockefeller Institute for Medical Research). By 1964 Zinder had become a full professor of genetics, and approximately 10 years later he was named John D. Rockefeller, Jr. Professor of Molecular Genetics. In 1993 Zinder was appointed dean of graduate and postgraduate studies.At Rockefeller, Zinder continued his studies of the molecular genetics of phages. He discovered the f2 phage, which was the first bacteriophage known to contain RNA as its genetic material, and demonstrated that RNA phage replication is not dependent on DNA (3).Zinder''s two Journal of Biological Chemistry (JBC) Classics reprinted here look at the phage f2 replicase. In the first paper, Zinder and his graduate student Nina V. Fedoroff show that the enzyme, purified on the basis of its poly(G) polymerase activity, could carry out the in vivo synthetic reactions involved in phage RNA replication. They also report that phage replicase activity is stimulated by salt and by a brief preliminary incubation at high ionic strength. The second paper, also by Zinder and Fedoroff and printed back-to-back with the first, compares the f2 poly(G) polymerase and replicase activities under a variety of conditions. They examined the effects of ionic strength, temperature, magnesium ion concentration, and template and substrate concentrations on the enzymes'' activities. Based on their results, Zinder and Fedoroff suggest a distinction between initiation and polymerization sites on the enzyme complex.Zinder remains at Rockefeller as John D. Rockefeller, Jr. Emeritus Professor and continues to research bacteriophage. Currently he is using genetics, biochemistry, and molecular biology to analyze the filamentous bacterial virus, f1, and its interactions with its host, Escherichia coli. His other studies relate to protein-DNA recognition, membrane anchoring, and questions of protein structure.In recognition of his many contributions to science, Zinder has received numerous honors and awards. These include the 1962 Eli Lilly Award in Microbiology and Immunology from the American Society of Microbiology, the 1966 Award in Molecular Biology from the National Academy of Sciences, the 1969 Medal of Excellence from Columbia University, and the 1982 Award for Scientific Freedom and Responsibility from the American Association for the Advancement of Science. Zinder became a member of the American Academy of Arts and Sciences in 1968 and of the National Academy of Sciences in 1969.Zinder''s coauthor on the two JBC papers also has gone on to a distinguished career in science. Fedoroff received her Ph.D. in 1972 and was a staff scientist at the Carnegie Institution of Washington. She joined the faculty of the Pennsylvania State University in 1995 and became the Evan Pugh Professor, Penn State''s highest academic honor, in 2002. She currently holds the Verne M. Willaman Chair of Life Sciences. In 2007, U. S. Secretary of State Condoleezza Rice named Fedoroff her science and technology adviser. She remains in this position today, serving U. S. Secretary of State Hillary Clinton. Fedoroff is a 2006 National Medal of Science laureate and a member of the National Academy of Sciences, the American Academy of Arts and Sciences, and the Phi Beta Kappa and Sigma Xi honor societies. Her current research focuses on the mechanisms that allow plants to withstand the environmental challenges of a changing climate.     

Structure-Function Relationships in Ribonuclease S: the Work of Frederic M. Richards

The Preparation of Subtilisin-modified Ribonuclease and the Separation of the Peptide and Protein Components(Richards, F. M., and Vithayathil, P. (1959) J. Biol. Chem. 234, 1459–1465)The Three-dimensional Structure of Ribonuclease-S. Interpretation of an Electron Density Map at a Nominal Resolution of 2 Å(Wyckoff, H. W., Tsernoglou, D., Hanson, A. W., Knox, J. R., Lee, B., and Richards, F. M. (1970) J. Biol. Chem. 245, 305–328)Frederic Middlebrook Richards (1925–2009) was born in New York City. He attended the Massachusetts Institute of Technology and, after a brief stint in the military, received his B.S. in 1948. Richards then enrolled in graduate school at Harvard Medical School, where he worked with Barbara Low and received his Ph.D. in 1952. After graduating he remained at Harvard for another year as a research fellow with Edwin Joseph Cohn, who was featured in a previous Journal of Biological Chemistry (JBC) Classic (1). Richards then moved to the Carlsberg Laboratory in Denmark where, with Kaj Linderstrøm-Lang and others, he began working on ribonuclease.Open in a separate windowFrederic M. RichardsAfter a short stint as a postdoctoral fellow at Cambridge University, Richards joined the faculty of the Department of Biochemistry at Yale University in 1955 as an assistant professor. He rose rapidly through the ranks, becoming professor in 1963. That year, Richards was also appointed chairman of the Department of Molecular Biology and Biophysics at Yale, which entailed a move from the Medical School to the Yale College campus. Following a sabbatical at Oxford University in 1967–1968, for which Richards and his wife Sally sailed their own boat with a small crew across the Atlantic Ocean, Yale merged the Medical School Department of Biochemistry and the Yale College Department of Molecular Biology and Biophysics to form a new university-wide Department of Molecular Biophysics & Biochemistry (MB&B) with Richards as its founding chair (1969–1973). Richards remained at Yale for his entire research career, eventually becoming Sterling Professor of Molecular Biophysics and Biochemistry.Much of Richard''s early research centered on bovine pancreatic ribonuclease (RNase). During his time at the Carlsberg laboratory, he showed that cleavage of RNase by the protease subtilisin produces a modified RNase (RNase S) that is still active (2). After starting his own lab at Yale, Richards was able to separate RNase S into a 20-residue S-peptide and a 102-residue S-protein, both of which lacked enzymatic activity. However, when the peptide and protein were recombined, the activity was recovered. Richards published an initial paper on this finding in 1958 (3). He followed this up with a more extensive article in the JBC, which is reprinted here as the first JBC Classic. In this paper, Richards and co-workers purified and characterized RNase S, separated it into S-peptide and S-protein, showed that almost all enzymatic activity is recovered when the two components are recombined, and also reported that the only observed change in covalent structure during the conversion of RNase A to RNase S is the hydrolysis of the peptide bond between residues 20 and 21.The demonstration that two separate, inactive fragments of the enzyme RNase A could be reconstituted to form an active enzyme provided the first experimental evidence that the ability of a protein to form a three-dimensional structure is an intrinsic property of its amino acid sequence. This work also foreshadowed the extensive RNase A refolding studies performed by Nobel laureate Christian Anfinsen, as discussed in a previous JBC Classic (4).In the 1960s Richards teamed up with Harold Wyckoff to solve the three-dimensional structure of RNase S. Initially, in 1967, they produced a 3.5 Å electron density map (5), which they used to determine the approximate conformation of the peptide chain. Three years later, they collected data to 2 Å, as reported in the second JBC Classic reprinted here. Using these data, Richards, Wyckoff, and colleagues produced an electron density map, which they used to determine the complete three-dimensional structure of RNase S. This structure tied with three others for the third protein structure ever solved to atomic resolution. Richards also showed that RNase S was enzymatically active in crystal form, putting to rest the widely held view at that time that protein crystal structures were irrelevant to the conformation and behavior of enzymes in solution.Richards received many honors and awards for his scientific achievements, including the Pfizer-Paul Lewis Award in Enzyme Chemistry (1965), election as Fellow of the American Academy of Arts and Sciences (1968), election to the National Academy of Sciences (1971), the Kai Linderstrøm-Lang Prize in Protein Chemistry (1978), the American Society for Biochemistry and Molecular Biology Merck Award (1988), the Stein and Moore Award of the Protein Society (1988), and the State of Connecticut Medal of Science (1995). He was also president of ASBMB (1979) and the Biophysical Society (1972–1973).     

The lipid A assembly pathway: the work of Christian Raetz

During his career, Christian Raetz has characterized many enzymes responsible for synthesizing or modifying lipid molecules, including the entire nine-enzyme pathway for the biosynthesis of lipid A, an essential part of bacterial outer membranes that plays a role in making many Gram-negative bacteria toxic. The findings from the two Journal of Biological Chemistry (JBC) Classic articles reprinted here were the start of Raetz'' elucidation of the enzymology, genetics, and structural biology of lipid A assembly.Fatty Acyl Derivatives of Glucosamine 1-Phosphate in Escherichia coli and Their Relation to Lipid A. Complete Structure of a Diacyl GlcN-1-P Found in a Phosphatidylglycerol-deficient Mutant (Takayama, K., Qureshi, N., Mascagni, P., Nashed, M. A., Anderson, L., and Raetz, C. R. H. (1983) J. Biol. Chem. 258, 7379–7385)The Biosynthesis of Gram-negative Endotoxin. Formation of Lipid A Precursors from UDP-GlcNAc in Extracts of Escherichia coli (Anderson, M. S., Bulawa, C. E., and Raetz, C. R. H. (1985) J. Biol. Chem. 260, 15536–15541)Christian Rudolf Hubert Raetz was born in 1946 in East Berlin. His parents were industrial chemists, and in the early 1950s the family moved to Ohio so his father could work for the Olin Mathieson Chemical Corporation. Being surrounded by chemists, Raetz naturally gravitated toward science and would do experiments with chemicals his father brought home from the lab. After receiving his bachelor''s degree in chemistry from Yale University in 1967, Raetz enrolled in a combined medical/doctoral program at Harvard Medical School. There, he worked with Eugene Kennedy, studying the enzymatic mechanism of phosphatidylserine synthesis in Escherichia coli and the role of liponucleotides in membrane biogenesis. Raetz graduated in 1973 and became a postdoctoral fellow at the National Institutes of Health, working with long-time Journal of Biological Chemistry (JBC) Editor-in-Chief Herbert Tabor at the National Institute of General Medical Sciences. (Tabor was featured in a previous JBC Classic (1).)Open in a separate windowChristian Raetz (left) and Eugene Kennedy at Kennedy''s 90th birthday symposium in 2009. Raetz is holding a biochemistry textbook written by Kennedy''s research advisor Albert Lehninger; thus the picture shows three generations of the Lehninger line of biochemists. Kennedy and Lehninger were featured in previous JBC Classics (3, 4).In 1976, Raetz joined the biochemistry department at the University of Wisconsin-Madison as an assistant professor and soon rose to the rank of full professor. At Madison, Raetz decided to combine genetics and lipid research, screening for mutants that formed defective cell membrane lipids in hopes of discovering the functions of structurally diverse lipid molecules.While studying Escherichia coli mutants with a defect in phosphatidylglycerophosphate synthetase (2), Raetz discovered a novel lipid building up in these mutants, the structure of which suggested that it might be a precursor to a membrane component known as lipid A. As reported in the first JBC Classic reprinted here, Raetz and his colleagues subjected the lipid to analysis by fast atom bombardment mass spectrometry and proton NMR spectroscopy and established its complete structure. The novel glucosamine-based lipid indeed turned out to be a key precursor of lipid A, and its discovery enabled Raetz to postulate testable hypotheses for lipid A biosynthesis. The discovery was also significant because lipid A anchors lipopolysaccharide (LPS) to the outer membrane of E. coli, and LPS, in turn, plays a role in making many Gram-negative bacteria, such as E. coli and Salmonella, toxic.The second JBC Classic reprinted here shows how the diacylated monosaccharide lipid A precursors are synthesized from known molecules by the fatty acylation of UDP-GlcNAc. From these results, Raetz postulated that the partitioning of UDP-GlcNAc between the lipid A pathway and peptidoglycan biosynthesis represents an important control point in the biogenesis of the Gram-negative envelope.Together, the findings from both JBC papers were the start of Raetz'' elucidation of the enzymology, genetics, and structural biology of lipid A assembly (see Fig. 1).Open in a separate windowFIGURE 1Raetz also discovered a large number of additional lipid A modification enzymes that are unique to certain subsets of Gram-negative bacteria but that can be reconstituted by heterologous expression in E. coli or Salmonella. Many of these enzymes are located on the periplasmic surface of the inner membrane or in the outer membrane, making them useful as reporters for lipid A trafficking. Some lipid A modification enzymes confer resistance to antimicrobial peptides, whereas others are important during pathogenesis.In 1987, Raetz left Madison to become executive director of biochemistry at Merck Research Laboratories and eventually became vice president of basic research, biochemistry, and microbiology at Merck. In addition to his own research, which was concerned with the identification and development of new antibiotics that target lipid A biosynthesis, Raetz supervised several ongoing lipid projects that included the final stages of simvastatin/Zocor (one of the first, and most successful, clinically and commercially, statin drugs produced by Merck) and finasteride/Proscar (used to treat benign prostatic hyperplasia and male pattern baldness).In 1993, Raetz returned to academia as chairman of biochemistry at the Duke University Medical Center and began focusing on the structural biology of the enzymes that make up the lipid A pathway. Today, Raetz remains at Duke where he is a George Barth Geller Professor of Biochemistry.In recognition of his contributions to science, Raetz has received many awards and honors including the 1979 Camille and Henry Dreyfus Teacher-Scholar Award, the 2002 American Society for Biochemistry and Molecular Biology Avanti Award in Lipids, the 2006 L. L. M. van Deenen Medal from the University of Utrecht, the 2006 Frederik B. Bang Award from the International Endotoxin and Innate Immunity Society, and election to the National Academy of Sciences in 2006.     

Mercury detoxification and natural product synthesis: the work of Christopher T. Walsh

Mercuric Reductase. Purification and Characterization of a Transposon-encoded Flavoprotein Containing an Oxidation-Reduction-active Disulfide (Fox, B., and Walsh, C. T. (1982) J. Biol. Chem. 257, 2498–2503)Cloning, Overproduction, and Characterization of the Escherichia coli Holo-acyl Carrier Protein Synthase (Lambalot, R. H., and Walsh, C. T. (1995) J. Biol. Chem. 270, 24658–24661)Christopher Thomas Walsh was born in 1944 in Boston, Massachusetts. He attended Harvard University, where he did undergraduate research with E. O. Wilson, publishing a first author paper on the composition of fire ant trail substance in Nature (1). He earned his A.B. in biology in 1965. Walsh then went to Rockefeller University to work with Leonard B. Spector, publishing six first author papers and earning a Ph.D. in 1970 with a dissertation titled “The Mechanism of Action of the Citrate Cleavage Enzyme.”Open in a separate windowChristopher T. WalshWalsh did a 2-year postdoctoral fellowship with Robert H. Abeles at Brandeis University before joining the faculty of the Massachusetts Institute of Technology (MIT) in 1972 as an assistant professor. He eventually became the Karl Taylor Compton Professor and chairman of the chemistry department there.Walsh''s initial research at MIT centered on studies of a class of enzyme inhibitors called “suicide substrates,” compounds that were not toxic to cells but resembled normal metabolites so closely that they underwent metabolic transformation to form products that were inhibitory. Walsh also started to explore novel chemical transformations in biology, which led to his elucidation of the process by which bacteria detoxify mercury-containing molecules in the environment by cleaving carbon-mercury bonds and then reducing the mercuric salt to elemental mercury. An enzyme that is central to this process is a flavoprotein called mercuric reductase. The enzyme catalyzes two-electron reduction of mercuric ions to elemental mercury using NADPH as an electron donor. The elemental mercury is volatile and is thus nonenzymatically removed from the environment.In the first Journal of Biological Chemistry (JBC) Classic reprinted here, Walsh and Barbara Fox describe the purification of mercuric reductase from Pseudomonas aeruginosa. To their surprise, they discovered that the enzyme had a high degree of similarity to lipoamide dehydrogenase and glutathione reductase, flavoenzymes that catalyze the transfer of electrons between pyridine nucleotides and disulfides. This paper initiated a series of studies investigating how the inorganic Hg²⁺ substrate is bound to two pairs of thiols, one in the active site and one as an exit site, and how electrons flow from NADPH through the FAD to the bound Hg²⁺.In 1987, Walsh moved to Harvard Medical School to learn more biology and medicine and to become chairman of the department of biological chemistry and molecular pharmacology. He continued to study biocatalysts and began exploring antibiotic and antitumor agents as well. One of his first major findings at Harvard explained the mechanism by which resistance develops to the antibiotic vancomycin (2), work that provided the foundation to create new antibiotics.Walsh also is widely recognized for spurring a renaissance in natural product biosynthesis. This started with his investigation of holo-acyl carrier protein synthase (ACPS), a phosphopantetheinyltransferase (PPTase) that transfers the 4′-phosphopantetheine (4′-PP) moiety from coenzyme A to Ser-36 of acyl carrier protein (ACP) in E. coli. Walsh and Ralph H. Lambalot purified ACPS to near homogeneity by exploiting the fact that ACPS could be refolded and reconstituted after elution from an apo-ACP affinity column under denaturing conditions. As reported in the second JBC Classic reprinted here, Walsh and Lambalot used N-terminal sequencing of ACPS to determine that dpj, an essential gene of previously unknown function, was the structural gene for ACPS. These studies led to the identification of other PPTase genes and enzymes involved in the conversion of apo forms of acyl and peptidyl carrier proteins in polyketide and nonribosomal peptide synthases/synthetases. This, in turn, allowed posttranslational activation of these multimodular enzymes when heterologously expressed in E. coli, which started Walsh on a 10-year, 200-paper focus on the characterization of the many enzymatic steps in assembly line biosynthesis of natural products.Currently, Walsh is the Hamilton Kuhn Professor of Biological Chemistry and Molecular Pharmacology at Harvard Medical School. He also was president of the Dana Farber Cancer Institute from 1992 to 1995. Walsh has received many honors and awards for his contributions to science. These include the Eli Lilly Award in Biochemistry (1979), the American Chemical Society (ACS) Arthur C. Cope Scholar Award in Organic Chemistry (1998), the ACS Repligen Award for Chemistry of Life Processes (1999), the ACS Alfred Bader Award for Bioorganic Chemistry (2003), the American Society for Microbiology Promega Biotechnology Research Award (2004), the American Society for Biochemistry and Molecular Biology Fritz Lipmann Award (2005), the ACS Murray Goodman Award (2007), and the Stanford University School of Medicine Pauling Medal and Lecture (2010). Walsh also was elected to the American Academy of Arts and Sciences (1988), the National Academy of Sciences (1989), and the American Philosophical Society (2003). He served on the JBC editorial board from 1978 to1980.¹     

Succeeding in Science Despite the Odds; Studying Metabolism with NMR by Mildred Cohn

A Study of Oxidative Phosphorylation with O¹⁸-labeled Inorganic Phosphate(Cohn, M. (1953) J. Biol. Chem. 201, 735–750)Nuclear Magnetic Resonance Spectra of Adenosine Di- and Triphosphate. II. Effect of Complexing with Divalent Metal Ions(Cohn, M., and Hughes, T. R. (1962) J. Biol. Chem. 237, 176–181)Mildred Cohn was born in New York City in 1913. When she was young, her father told her she could achieve anything she chose to, but not without some difficulty because she was both female and Jewish. With her parents'' encouragement, Cohn moved rapidly through the New York public school system and graduated from high school at age 14. She decided to go to Hunter College in Manhattan, then an all-girls college, and majored in chemistry and minored in physics. Hunter''s attitude toward science education at that time can be summed up by the chairman of the chemistry department who declared that it was not ladylike for women to be chemists and that his sole purpose was to prepare his students to become chemistry teachers.When Cohn graduated from Hunter College in 1931 she tried to get a scholarship for graduate studies in chemistry but was unsuccessful. She enrolled in Columbia University nonetheless and used her savings to pay for her education. At Columbia, she studied under Nobel laureate Harold Urey but had to drop out after a year because of lack of money. She then took a job with the National Advisory Committee of Aeronautics and after a few years was able to earn enough money to return to Columbia. Working with Urey, she studied ways of separating different isotopes of carbon and received her Ph.D. in physical chemistry in 1937.Unfortunately, jobs were scarce in 1938, during the years of the Great Depression, and academic positions for women were even more scarce. Industrial recruiters regularly posted notices announcing that, “Mr. X of Y Company will interview prospective doctorate recipients—Male, Christian”(1).¹ With Urey''s help, Cohn was able to obtain a postdoctoral position at George Washington University with future Nobel prize winner Vincent du Vigneaud. In du Vigneaud''s laboratory, Cohn pioneered the effort to use isotopic tracers to follow the metabolism of sulfur-containing compounds, the subject of a previous Journal of Biological Chemistry (JBC) Classic (2). Cohn worked with du Vigneaud for 9 years and moved with him to New York when he went to Cornell Medical College.In 1946, Cohn went to Washington University in St. Louis to work with Carl and Gerty Cori, Nobel prize laureates and authors of a previous JBC Classic (3), who were studying biological catalysts. There, she did independent research, mainly focusing on using isotopes and NMR to study metabolic processes. Cohn was promoted to Associate Professor in Biochemistry in 1958 but left Washington University 2 years later to move to the University of Pennsylvania School of Medicine. She became a full Professor in 1961 and retired as Benjamin Rush Professor Emerita of Biochemistry and Biophysics in 1982.Once, when asked what her most exciting scientific moments were (4), Cohn replied, “In 1958, using nuclear magnetic resonance, I saw the first three peaks of ATP (5). That was exciting. [I could] distinguish the three phosphorus atoms of ATP with a spectroscopic method, which had never been done before. Another paper, in 1962 (the second JBC Classic reprinted here), was about the effect of metal ions on the phosphorus spectrum of ATP. And earlier, I found that oxygen in inorganic phosphate exchanged with water through oxidative phosphorylation (the first JBC Classic reprinted here).”Cohn''s study of oxidative phosphorylation came at a time when it was known that phosphorylation occurred concomitantly with oxidation in the electron transport chain. However, no one had yet discovered the nature of the interaction of the electron transport system with phosphate or any part of the phosphorylating system. Cohn approached this problem by tracking the loss of O¹⁸ from inorganic phosphate during oxidative phosphorylation in rat liver mitochondria. In the first JBC Classic reprinted here, she describes her findings as, “a new reaction which occurs in oxidative phosphorylation associated with the electron transport system has been observed in the rat liver mitochondria with α-ketoglutarate, â-hydroxybutyrate, and succinate as substrates. This reaction manifests itself by a replacement of O¹⁸ with normal O¹⁶ in inorganic phosphate labeled with O¹⁸ and parallels the phosphorylation which is associated with the oxidation.” Cohn concluded that water must be involved in this reaction because there was no other source of oxygen large enough to account for the amounts she saw introduced into inorganic phosphate. In the second JBC Classic Cohn describes her use of NMR to examine the structural changes in ADP and ATP caused by various divalent metal ions. Cohn knew that divalent ions were involved in enzymatic reactions of ADP and ATP but didn''t know their functions. Using NMR, she measured the changes in the chemical shifts in the peaks of the ATP and ADP phosphorus nuclei in the presence of Mg²⁺, Ca²⁺, and Zn²⁺ as well as the paramagnetic ions Cu²⁺, Mn²⁺, and Co²⁺. By analyzing the resultant spectra, she was able to determine which metals bound to which phosphate groups and thus gained insight into the nature of the metal complexes formed.Cohn received many awards and honors for her contributions to science, including the National Medal of Science in 1982, “for pioneering the use of stable isotopic tracers and nuclear magnetic resonance spectroscopy in the study of the mechanisms of enzymatic catalysis,” as well as election to the National Academies of Science in 1971. She also served the American Society of Biological Chemists (ASBC), now American Society for Biochemistry and Molecular Biology (ASBMB), in many ways. She was President of the Society in 1978 and was on the Federation of American Societies for Experimental Biology (FASEB) Board from 1978 to 1980 as ASBC representative. In addition, Cohn was the first woman to be appointed to the JBC Editorial Board.Despite her success, Cohn''s father was right about the difficulties she would encounter in her life. “My career has been affected at every stage by the fact that I am a woman, beginning with my undergraduate education, which was very inferior in chemistry, and physics was not even offered [as a major] at Hunter College, unlike the excellent science education that my male counterparts received at City College,” she notes. “In my day, I experienced discrimination in academia, government, and industry.”     

Biotin-dependent Enzymes: the Work of Feodor Lynen

The Enzymatic Synthesis of Holotranscarboxylase from Apotranscarboxylase and (+)-Biotin. I. Purification of the Apoenzyme and Synthetase; Characteristics of the Reaction(Lane, M. D., Young, D. L., and Lynen, F. (1964)J. Biol. Chem.239, 2858–2864)Feodor Felix Konrad Lynen (1911–1979) was born in Munich, Germany. He was undecided about his career during his early education and even considered becoming a ski instructor. Ultimately, he enrolled in the Department of Chemistry at the University of Munich where he studied with Nobel laureate Heinrich Wieland and received his doctorate degree in 1937. Three months later, he married Wieland''s daughter, Eva.Open in a separate windowFeodor LynenAfter graduating, Lynen remained at Munich University as a postdoctoral fellow. He was appointed lecturer in 1942 and assistant professor in 1947. When World War II broke out, Lynen was exempt from military service because of a knee injury resulting from a ski accident in 1932. However, the war made it difficult to continue to do research in Munich, and Lynen moved his laboratory to the small village of Schondorf on the Ammersee. This was lucky because in 1945 Munich University''s Department of Chemistry was destroyed. Lynen continued his work at various laboratory facilities and eventually returned to the rebuilt Department of Chemistry in 1949.During the 1940s, Lynen began studying the biosynthesis of sterols and lipids. He eventually initiated a collaboration with Konrad Bloch, whose cholesterol research was featured in a previous Journal of Biological Chemistry (JBC) Classic (1). Working together, Bloch and Lynen were able to elucidate the steps in the biosynthesis of cholesterol. An especially significant finding made by Lynen was that acetyl coenzyme A (previously discovered by JBC Classic author Fritz Lipmann (2)) was essential for the first step of cholesterol biosynthesis. Lynen later determined the structure of acetyl-CoA. This work on cholesterol resulted in Bloch and Lynen being awarded the 1964 Nobel Prize in Physiology or Medicine.In 1953, Lynen was made full professor at the University of Munich. A year later, he was named director of the newly established Max Planck Institute for Cell Chemistry. He continued to work on fats but also turned his focus to biotin-dependent enzymes. In 1962, he was joined by JBC Classic author M. Daniel Lane (3), who had come to Munich to work with Lynen on a sabbatical leave. Lane was studying the biotin-dependent propionyl-CoA carboxylase and had previously determined that its biotin prosthetic group was linked to the enzyme through an amide linkage to a lysyl ϵ-amino group.Before leaving for Munich, Lane developed an apoenzyme system with which to investigate the mechanism by which biotin became attached to propionyl-CoA carboxylase. This system made use of Propionibacterium shermanii, which expressed huge amounts of methylmalonyl-CoA:pyruvate transcarboxylase, another biotin-dependent enzyme. The organism also had an absolute requirement for biotin in its growth medium and produced large amounts of the apotranscarboxylase when grown at very low levels of biotin.As reported in the JBC Classic reprinted here, Lane and Lynen were able to resolve and purify both the apotranscarboxylase and the synthetase that catalyzed biotin loading onto the apoenzyme. Dave Young, a postdoctoral fellow who had recently completed his medical training at Duke University, collaborated with them on these studies. In a second paper reprinted in the Lane Classic (4), Lane and Lynen showed that the synthetase catalyzed a two-step reaction. The first step involved the ATP-dependent formation of biotinyl-5′-AMP and pyrophosphate after which the biotinyl group was transferred from the AMP derivative to the appropriate lysyl ϵ-amino group of the apotranscarboxylase. Lane and Lynen also showed that the covalently bound biotinyl prosthetic group, like free biotin, was carboxylated on the 1′-N position (5).In 1972, Lynen moved to the recently founded Max Planck Institute for Biochemistry. Between 1974 and 1976, he was acting director of the Institute while continuing to oversee a lab at the University of Munich. He remained at the Institute until his death in 1979.In addition to the Nobel Prize, Lynen received many honors and awards. These include the Neuberg Medal of the American Society of European Chemists and Pharmacists (1954), the Liebig Commemorative Medal of the Gesellschaft Deutscher Chemiker (1955), the Carus Medal of the Deutsche Akademie der Naturforscher Leopoldina (1961), and the Otto Warburg Medal of the Gesellschaft für Physiologische Chemie (1963).     

From DNA enzymes to cone snail venom: the work of Baldomero M. Olivera

Processivity of DNA Exonucleases (Thomas, K. R., and Olivera, B. M. (1978) J. Biol. Chem. 253, 424–429)Neuronal Calcium Channel Inhibitors. Synthesis of ω-Conotoxin GVIA and Effects on ⁴⁵Ca Uptake by Synaptosomes (Rivier, J., Galyean, R., Gray, W. R., Azimi-Zonooz, A., McIntosh, J. M., Cruz, L. J., and Olivera, B. M. (1987) J. Biol. Chem. 262, 1194–1198)The two papers being recognized here as JBC Classics speak to the journeys Baldomero “Toto” Olivera at the University of Utah has made in his life. A director of a program project funded by the National Institute of General Medical Sciences and a professor at the Howard Hughes Medical Institute, Olivera''s papers highlight how doing research in two different countries ultimately influenced his focus and contributions to molecular biology and biochemistry.Open in a separate windowBaldomero “Toto” Olivera. Photo courtesy of Olivera.Olivera began his career as a DNA biophysical chemist and enzymologist. He arrived in the United States in the 1960s to do his graduate work at the California Institute of Technology after completing a bachelor''s degree in chemistry in the Philippines. He joined the laboratory of Norman Davidson to study the biophysical chemistry of DNA. When Olivera was ready to graduate with his Ph.D. degree, Davidson suggested that Olivera go to I. Robert Lehman''s laboratory at Stanford University for his postdoctoral training. “He knew it was my intention to return to the Philippines,” recalls Olivera. Davidson felt it would be easier for Olivera to study DNA enzymology, rather than biophysical chemistry, in a Philippine academic setting because the field did not necessarily demand expensive and sophisticated instrumentation.Olivera followed his thesis advisor''s suggestion and, as a result, became an expert in DNA enzymology, including exonucleases, a large class of DNA-degrading enzymes. The first JBC paper recognized here as a Classic was published in 1978 as Olivera was starting out as an independent researcher. In it, Olivera and his first graduate student, Kirk Thomas, investigated whether or not exonuclease I, first discovered in Escherichia coli by Lehman, and other exonucleases of E. coli were processive. This was at a time when little was known about nucleic acid enzymes: restriction enzymes were just starting to gain traction, and genome sequencing was far from reality. Olivera explains that no one had given much thought to how exonucleases functioned. “The significance of this paper was that it showed that the enzymes that we examined were very different using a new parameter processivity that had never been assessed for exonucleases,” he says.Olivera and Thomas designed an assay that was based on a synthetic nucleic acid chain that contained ³H on one end and ³²P on the other. Researchers knew that exonucleases selected either the 5′- or 3′-end of the DNA to start chewing. The rationale of the Thomas and Olivera assay was that if the enzyme dissociated after every single catalytic event, one label, either the ³H or ³²P, would come off the polymer. However, if the enzyme clung to the polymer and kept chewing until the whole polymer was degraded, both radioactive labels would appear simultaneously in solution.Open in a separate windowOlivera with his first graduate student, Kirk Thomas. Photo courtesy of Olivera.Thomas and Olivera demonstrated that of the eight exonucleases they tested, only the E. coli exonuclease I and λ-exonuclease were processive, meaning that once they got started, they kept on cutting the same piece of DNA before dissociating. The others, such as the spleen and T7 exonucleases, were not processive and frequently came off the DNA.Lehman explains that at the time of this JBC paper, “methods had not yet been developed to measure quantitatively the processivity of either a DNA polymerase or a DNA exonuclease. Their paper made an important contribution to the field of DNA enzymology by describing for the first time a quantitative method for doing so and applied it to eight different DNA exonucleases, an enzymological tour de force.”The second paper highlighted as a JBC Classic was published ten years later and shows a shift in Olivera''s career. The article concerns the synthesis of a peptide found in the venom of the cone snail Conus geographus, which is indigenous to the Indo-Pacific region. All 700 types of cone snails have a special tooth that they use like a harpoon. A venom gland attached to the tooth releases the poisonous peptides to paralyze or even kill prey. These snails have to be handled with great care or not handled at all. Some can sting and cause pain like bees, but C. geographus can kill humans when it stings.There is no scientific connection between DNA enzymes and snail venom. Olivera explains that when he had returned to the Philippines as an assistant professor in the College of Medicine at the University of the Philippines, his laboratory “had absolutely no equipment. It was clear I wasn''t going to be very competitive in DNA replication [research], so we decided we''d find a project that we could start without any equipment. I collected shells as a kid, so I knew about this particular snail that killed people. I had purified enzymes as a post-doc and figured I could purify toxins by injecting them into mice, which didn''t require any equipment at all.”Olivera''s group was soon isolating and characterizing peptides from the cone snail venom. The peptides are known as conotoxins. In doing so, Olivera established the field of conotoxin research, which had a significant impact on fundamental research and medicine. For example, a peptide isolated by Olivera''s group has been approved as a drug for severe pain that cannot be relieved by morphine.Olivera had part-time appointments in the United States while maintaining his full-time position in the Philippines. He first began as a visiting associate professor at Kansas State University and later at the University at Utah. “I would spend seven or eight months in the Philippines and five or four months in the U.S,” he says. Olivera became a full-time member of the faculty at the University of Utah in the 1970s after political and economic upheaval in the Philippines over Ferdinand Marcos'' rule made Olivera decide to return full-time to the United States.Open in a separate windowConus snails. Photo courtesy of Olivera.Open in a separate windowConus snail attacks a fish. Photo courtesy of Olivera.The toxins made by the Conus snails are highly specific for particular targets in the nervous system, such as ion channels. For example, the μ-conotoxins hit sodium receptor ion channels, and ω-conotoxins (one of which, ω-GVIA, is described in this JBC Classic) bind to neuronal calcium channels to inhibit calcium uptake at the presynaptic junction and shut down biochemical signaling at certain synapses.ω-Conotoxin GVIA is a 27-amino acid peptide originally called the “shaker” peptide because it made mice shake. “A number of physiological experiments were done to suggest that it acted at synapses, potentially on calcium channels,” says Olivera. “The importance of this paper is that for the first time the peptide was chemically synthesized and became available to the whole neuroscience community.”The neuroscience community desperately needed this peptide. Up to this point, neuroscientists relied on dihydropyridines to study voltage-gated calcium channels. However, these dihydropyridines had confusing effects on neuronal voltage-gated calcium channels, which made data interpretation difficult. With ω-conotoxin GVIA as a synthetic peptide, neuroscientists now had a molecular tool that clearly targeted a very specific type of neuronal voltage-gated calcium channel.The peptide was short enough to be amenable to synthesis, and Olivera is grateful to his collaborator, Jean Rivier, who was an expert in synthesizing neuropeptides, for the successful synthesis of this peptide. The peptide had only 27 amino acids but contained three disulfide bonds, “so there were fifteen possible isomers,” recalls Olivera. “You had to get the cross-linking right to end up with the biologically active isomer.”The advantage was that Olivera and colleagues had purified the native peptide, so they could compare their synthesis attempts with the native molecule. “At the beginning, we didn''t even know what the true disulfide bonding was, so we did the work qualitatively to just show the synthetic material and native material co-eluted in a column.” The investigators later established how the disulfide bonds were arranged. Rivier, Olivera, and the rest of the team went on to show that their synthetic peptide behaved just like the natural one in inhibiting calcium entry at chicken synaptosomes and was biologically active.John Exton at Vanderbilt University says “The conotoxins have proved to be extremely important molecular probes in neuroscience in defining functional roles for many receptors and ion channels.”When the paper was published, Olivera was deluged with requests for the peptide. Rivier had been able to synthesize a sizeable amount, and because it was active at subpicomolar concentrations, a little bit of it went a long way. Olivera was able to distribute the peptide, and eventually, several commercial enterprises got into the business of producing and supplying it.“I believe there is something on the order of two thousand studies in the literature using this particular peptide,” says Olivera. “It''s interesting that there are hundreds of thousands of peptides in Conus venom that we call conotoxins. But among physiologists, if you say conotoxin, this is the peptide they think of because this is the one that''s most widely used.” In fact, points out Olivera, when the neuronal calcium channel was purified eight years later, it was actually called the conotoxin receptor.     

The Use of Isotope Effects to Study Dopamine ��-Monooxygenase Catalysis: the Work of Judith P. Klinman

Deduction of the Kinetic Mechanism in Multisubstrate Enzyme Reactions from Tritium Isotope Effects. Application to Dopamine β-Hydroxylase(Klinman, J. P., Humphries, H., and Voet, J. G. (1980) J. Biol. Chem. 255, 11648–11651)Use of Isotope Effects to Characterize Intermediates in the Mechanism-based Inactivation of Dopamine β-Monooxygenase by β-Chlorophenethylamine(Bossard, M. J., and Klinman, J. P. (1990) J. Biol. Chem. 265, 5640–5647)Judith Klinman was born Judith Pollock in 1941 in Philadelphia, Pennsylvania. Early on, she realized she was interested in science and decided to go to the University of Pennsylvania to study chemistry. However, her parents wanted her to become a medical technologist and forget about science. She eventually got them to agree to let her go if she got a scholarship; she graduated in 1962 with an A.B. She then decided to go to graduate school at New York University, but moved back to Penn after a year. Working with Edward R. Thornton, she completed her Ph.D. in 3 years, publishing a thesis titled “A Kinetic Study of the Hydrolysis and Imidazole-catalyzed Hydrolysis of Substituted Benzoyl Imidazole in Light and Heavy Water.”Open in a separate windowJudith P. KlinmanAfter graduating in 1966, Klinman first carried out postdoctoral research with David Samuel at the Weizmann Institute of Science in Israel and later with Journal of Biological Chemistry (JBC) Classic author Irwin Rose (1) at the Institute for Cancer Research in Philadelphia. Returning to the United States in 1968, she joined the Institute for Cancer Research in Philadelphia, where she was a research scientist for 10 years. In 1978 she became the first woman professor in the chemistry department of the University of California, Berkeley, where she continues to do research today as Professor of Chemistry and Molecular and Cell Biology in the Department of Chemistry.Throughout her research career, Klinman has contributed extensively to the understanding of the fundamental properties that underlie enzyme catalysis. Early in her career, she developed the application of kinetic isotope effects to the study of enzyme catalysis, showing how these probes can be used to uncover chemical steps, to determine kinetic order, and to obtain substrate dissociation constants. The two JBC Classics reprinted here stem from her use of isotope effects to isolate the chemical steps involved in the dopamine β-monooxygenase-catalyzed conversion of dopamine and oxygen to norepinephrine and water.In 1965, JBC Classic author Seymour Kaufman (2) suggested that oxygen binding precedes the addition of substrate in dopamine β-hydroxylase (now called dopamine β-monooxygenase) (3). However, confirmation of this hypothesis was hard to do using classical methods such as product and dead-end inhibition studies and equilibrium exchange techniques because of the apparent reversibility of the chemical step and the fact that one of the reaction products was water. In the first Classic, Klinman was able to disprove this hypothesis and use the sensitivity of kinetic tritium isotope effects to changes in oxygen concentrations in the reaction to provide unequivocal evidence for a random order of addition of dopamine and oxygen to dopamine β-hydroxylase.In the second Classic, Klinman uses isotope effects to study the inhibition of dopamine β-monooxygenase by β-chlorophenethylamine. Previously, she had postulated an inhibition mechanism in which bound α-aminoacetophenone was generated followed by an intramolecular redox reaction to yield a ketone-derived radical cation as the inhibitory species (4). However, she was unable to determine whether inhibition by α-aminoacetophenone occurred at the reductant- or substrate-binding site and was unable to provide evidence of keto-enol tautomerization of α-aminoacetophenone at the active site. Both of these questions were addressed using kinetic isotope effects. As reported in the JBC Classic, she showed that α-aminoacetophenone acts at the substrate-binding site and that there are two isotope-sensitive steps in β-chlorophenethylamine inactivation, with the second step attributed to an isotope-sensitive partitioning of the bound enol of α-aminoacetophenone between reketonization and oxidation.Over the years, Klinman continued her investigations into enzyme catalysis. In 1990 she demonstrated the presence of the neurotoxin, 6-hydroxydopaquinone (TPQ), at the active site of a copper-containing amine oxidase from bovine plasma, overcoming years of incorrect speculation regarding the nature of the active site structure and opening up the currently active field of protein-derived cofactors. Subsequent work from her group showed that the extracellular protein lysyl oxidase, responsible for collagen and elastin cross-linking, contains a lysine cross-linked variant of TPQ. Since the 1990s, Klinman''s kinetic studies of enzyme reactions have demonstrated anomalies that implicate quantum mechanical hydrogen tunneling in enzyme-catalyzed hydrogen activation reactions. In recent years she has developed a unique set of experimental probes for determining the mechanism of oxygen activation. These probes are beginning to shed light on how proteins can reductively activate O₂ to free radical intermediates, while avoiding oxidative damage to themselves.In addition to being the first woman faculty member in the physical sciences at the University of California, Berkeley, Klinman was the first woman Chair of the Department of Chemistry from 2000 to 2003. During her tenure at Berkeley she has been a Chancellor''s Professor, Guggenheim Fellow, and Miller Fellow. She was elected to the National Academy of Sciences (1994), the American Academy of Arts and Sciences (1993), and the American Philosophical Society (2001) and has received the Repligen Award (1994) and the Remsen Award (2005) from the American Chemical Society and the Merck Award from the American Society for Biochemistry and Molecular Biology (2007). Klinman was also President of the American Society for Biochemistry and Molecular Biology in 1998 and served on the editorial board of the Journal of Biological Chemistry from 1979 to 1984.