The Mechanism of Amino Acid Activation: the Work of Mahlon Hoagland

Enzymatic Carboxyl Activation of Amino Acids(Hoagland, M. B., Keller, E. B., and Zamecnik, P. C. (1956) J. Biol. Chem. 218, 345–358)Mahlon Bush Hoagland was born in Boston, Massachusetts in 1921. He attended Harvard University and graduated in 1943. Knowing that he wanted to be a surgeon, Hoagland then enrolled at Harvard Medical School. However, he was diagnosed with tuberculosis, and his poor health prevented him from becoming a surgeon when he received his M.D. in 1948. Instead, he accepted a research position at Massachusetts General Hospital. In 1953, he became a postdoctoral fellow with Journal of Biological Chemistry (JBC) Classic author Fritz Lipmann (1) at Huntington Laboratories (also at Massachusetts General Hospital), and a year later, he moved to an adjoining laboratory to work on protein synthesis with JBC Classic author Paul Zamecnik (2).Open in a separate windowMahlon HoaglandInspired by Lipmann''s insights into acyl activation mechanisms, Hoagland used a cell-free system created by Zamecnik that carried out net peptide bond formation using ¹⁴C-amino acids (3) to uncover the mechanism of amino acid activation. As reported in the JBC Classic reprinted here, he isolated an enzyme fraction that, in the presence of ATP and amino acids, catalyzed the first step in protein synthesis: the formation of aminoacyl adenylates or activated amino acids. Using data from analysis of this fraction, Hoagland presented a scheme for amino acid activation in his Classic paper.A few years later, Zamecnik and Hoagland discovered a molecule that is essential for protein synthesis: tRNA. This discovery is the subject of the Zamecnik Classic (2).After the discovery of tRNA, Hoagland spent the next year (1957–1958) at Cambridge University''s Cavendish laboratories working with Francis Crick. During that year he traveled to France to visit the Institute Pasteur in Paris. Experiments begun at the Institute would, by 1960, lead to the discovery of messenger RNA (mRNA).When he returned to the United States, Hoagland was appointed associate professor of microbiology at Harvard Medical School. He remained there until 1967 when he accepted a position as professor at Dartmouth Medical School. In 1970, he became the director of the Worcester Foundation for Experimental Biology, a Massachusetts research institute founded by his father. He retired in 1985 and currently lives in Thetford, Vermont.Hoagland has received several awards and honors in recognition of his contributions to science. These include the 1976 Franklin Medal, the 1982 and 1996 Book Awards from the American Medical Writers Association, and membership in the American Academy of Arts and Sciences and the National Academy of Sciences.     

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).     

Bacterial chimeras and reversible phosphorylation: the work of Walther Stoeckenius

In 1971, Walther Stoeckenius discovered that Halobacterium halobium contains a purple pigment that is chemically similar to rhodopsin and works as a light-driven proton pump. This discovery set Stoeckenius on a research path centered on bacteriorhodopsin, which included the creation of a bovine-soybean-halobacteria chimera that was able to produce ATP when exposed to light and the discovery of a class of proteins that are phosphorylated in a light-dependent manner.Reconstitution of Purple Membrane Vesicles Catalyzing Light-driven Proton Uptake and Adenosine Triphosphate Formation (Racker, E., and Stoeckenius, W. (1974) J. Biol. Chem. 249, 662–663)Light-regulated Retinal-dependent Reversible Phosphorylation of Halobacterium Proteins (Spudich, J. L., and Stoeckenius, W. (1980) J. Biol. Chem. 255, 5501–5503)Walther Stoeckenius was born in 1921 in Giessen, Germany. He earned an M.D. degree from the University of Hamburg in 1950, after which he spent 18 months doing clinical work as an intern. In 1952, he began postdoctoral work at the Institute for Tropical Medicine in Hamburg, using electron microscopy to study the development of pox viruses. Two years later, he joined the Department of Pathology at the University of Hamburg as an assistant professor and became Docent for Pathology in 1958. At Hamburg, Stoeckenius continued to use electron microscopy to explore the fine structure of cells and the lipid membrane.In 1959, Stoeckenius left Germany to become a research associate in Keith Porter''s laboratory at Rockefeller University. After a few months, he became an assistant professor at Rockefeller, remaining there for 8 years and eventually becoming an associate professor. He continued to work on membrane structure, studying Halobacterium halobium, until he accepted a professorship at the University of California, San Francisco in 1967.In San Francisco, Stoeckenius focused more on biochemical techniques rather than electron microscopy. In collaboration with Dieter Oesterhelt, he discovered that H. halobium contains a purple pigment (bacteriorhodopsin) that is chemically similar to rhodopsin (1) and plays an important role in light energy storage in halobacteria, working as a light-driven proton pump (2).This discovery led to a collaboration with Journal of Biological Chemistry (JBC) Classic author Efraim Racker (3) in which Stoeckenius and Racker created a thoroughly unnatural vesicle. As reported in the first JBC Classic reprinted here, they used sonication to recombine membrane lipids from soybeans, bacteriorhodopsin from halobacteria, and ATPase from beef mitochondria. The resulting artificial vesicles were able to produce ATP when exposed to light. The chimeric vesicles also formed a simple model system for a biological proton pump capable of generating ATP from ADP and P_i.Stoeckenius continued to study bacteriorhodopsin and its light-driven proton uptake in bacteria. As reported in the second JBC Classic reprinted here, he discovered that phosphorylation is regulated by light absorbed by bacteriorhodopsin (4). Using [³²P]orthophosphate pulse labeling, Stoeckenius and John Spudich identified a class of phosphoproteins in H. halobium. Exposing labeled whole cells to light resulted in rapid dephosphorylation of two of the proteins, which were rapidly rephosphorylated upon darkening of the cells. The light sensitivity of the proteins was responsive to the presence of retinal, indicating that the dephosphorylation depended on rhodopsin-like (retinal-containing) photoreceptors.Stoeckenius currently is Professor Emeritus in the Department of Biochemistry and Biophysics and the Cardiovascular Research Institute at the University of California, San Francisco. He was elected to the National Academy of Sciences in 1978.     

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     

Yasutomi Nishizuka's Discovery of Protein Kinase C

Studies on a Cyclic Nucleotide-independent Protein Kinase and Its Proenzyme in Mammalian Tissues. I. Purification and Characterization of an Active Enzyme from Bovine Cerebellum (Takai, Y., Kishimoto, A., Inoue, M., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7603–7609)Direct Activation of Calcium-activated, Phospholipid-dependent Protein Kinase by Tumor-promoting Phorbol Esters (Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U., and Nishizuka, Y. (1982) J. Biol. Chem. 257, 7847–7851)Yasutomi Nishizuka (1932–2004) was born in Ashiya-city, Japan. He attended Kyoto University and obtained his M.D. in 1957 and his Ph.D. in 1962, working with Journal of Biological Chemistry (JBC) Classic author Osamu Hayaishi (1). He then spent a year as a postdoctoral fellow at Rockefeller University with Fritz Lipmann (also featured in a JBC Classic (2)) before returning to Kyoto University to resume work with Hayaishi. During this time, Nishizuka studied the biosynthesis of nicotinamide adenine dinucleotide (NAD), the involvement of GTP in ribosomal protein translation, and ADP-ribosylation by diphtheria toxin.Open in a separate windowYasutomi NishizukaIn 1969, Nishizuka accepted the position of full professor and head of the department of biochemistry at the Kobe University School of Medicine. There, Nishizuka became interested in the role of protein kinases in the regulation of cell functions. This led to his discovery of a novel protein kinase, which he published in the first paper reprinted here as the JBC Classic. As Nishizuka reported in that paper, he and his colleagues partially purified the kinase from bovine cerebellum. They found that the enzyme was capable of phosphorylating histone and protamine and that it probably was produced from its precursor protein by a limited proteolytic reaction. The detailed properties of the proenzyme and its conversion to active protein kinase were reported in a subsequent JBC paper (3). Nishizuka named this new enzyme “protein kinase C (PKC).”A paper published by Nishizuka two years later in the JBC (4) showed that PKC was activated without limited proteolysis by a membrane-associated factor in the presence of a low concentration of Ca²⁺. In 1980, he published another paper in the JBC (5) showing that the membrane-associated factor was diacylglycerol, which suggested that the lipid could be a novel second messenger generated by receptor-stimulated phosphoinositide hydrolysis. Nishizuka validated this idea by showing that treating platelets with a combination of a Ca²⁺ ionophore and membrane-permeant short chain diacylglycerol mimicked stimulation by the aggregating agent thrombin (6). This discovery was a major advance in the understanding of cell signaling.Nishizuka and his colleagues then discovered that PKC is the biological target of tumor-promoting phorbol esters. At that time, it was well known that croton oil augmented carcinogenesis when it was applied at weekly intervals to the skin of mice in conjunction with a very dilute solution of benz[a]pyrene in acetone. The oil contained phorbol ester, a powerful tumor promoter, and caused a wide variety of cellular responses that were similar to those seen with hormones. Nishizuka speculated that the phorbol ester was producing diacylglycerol to activate PKC. However, upon further investigation, he realized that the phorbol ester contained a diacylglycerol-like structure and thus might activate PKC directly. In a series of experiments, published in the second JBC Classic reprinted here, Nishizuka was able to show that the phorbol ester activated PKC directly. This discovery showed that PKC was important for cell proliferation and cancer. It also established the use of phorbol esters as crucial tools for the manipulation of PKC activation in intact cells, eventually allowing the elucidation of the wide range of cellular processes regulated by this enzyme.This research laid the foundation for an enormous number of studies on the complex PKC family, many of them from Nishizuka''s group.In 1975, Nishizuka became president of the University of Kobe, a position that he held until 2001. He received numerous awards and honors for his research, including the Gairdner Foundation International Award (1988), the Alfred P. Sloan Jr. Prize (1988), the Japan Order of Culture (1988), the Albert Lasker Basic Medical Research Award (1989), the Kyoto Prize (1992), the Wolf Prize in Medicine (1995), the Jimenez Diaz Award (1995), and the Schering Prize (1995). He also served as a foreign member and honorary fellow of various academies, including the National Academies of Science, the Royal Society, l''Academie des Sciences, die Deutsche Akademie der Naturforscher Leopoldina, le Real Academia de Ciencias, the Asiatic Society, and the Japan Academy.¹     

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.     

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     

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.     

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.     

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).     

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.     

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.     

Leucine: tRNA Ligase from Cultured Cells of Nicotiana tabacum var. Xanthi: Evidence for de Novo Synthesis and for Loss of Functional Enzyme Molecules

Leucine:tRNA ligase was assayed in extracts from cultured tobacco (Nicotiana tabacum) XD cells by measuring the initial rate of aminoacylation of transfer RNA with l-[4,5-³H]leucine. Transfer RNA was purified from tobacco XD cells after the method of Vanderhoef et al. (Phytochemistry 9: 2291-2304). The buoyant density of leucine:tRNA ligase from cells grown for 100 generations in 2.5 mm [¹⁵N]nitrate and 30% deuterium oxide was 1.3397. After transfer of cells into light medium (2.5 mm [¹⁴N]nitrate and 100% H₂O) the ligase activity increased and the buoyant density decreased with time to 1.3174 at 72 hours after transfer. It was concluded that leucine:tRNA ligase molecules were synthesized de novo from light amino acids during the period of activity increase. The width at half-peak height of the enzyme distribution profiles following isopycnic equilibrium centrifugation in caesium chloride remained constant at all times after transfer into light medium providing evidence for the loss of preexisting functional ligase molecules. It was concluded that during the period of activity increase the cellular level of enzyme activity was determined by a balance between de novo synthesis and the loss of functional enzyme molecules due to either inactivation or degradation.