Expression of the Streptococcus pneumoniae type 3 synthase in Escherichia coli. Assembly of type 3 polysaccharide on a lipid primer.

Synthesis of the type 3 capsular polysaccharide of Streptococcus pneumoniae is catalyzed by the membrane-localized type 3 synthase, which utilizes UDP-Glc and UDP-GlcUA to form high molecular mass [3-beta-d-GlcUA-(1-->4)-beta-d-Glc-(1-->](n). Expression of the synthase in Escherichia coli resulted in synthesis of a 40-kDa protein that was reactive with antibody directed against the C terminus of the synthase and was the same size as the native enzyme. Membranes isolated from E. coli contained active synthase, as demonstrated by the ability to incorporate Glc and GlcUA into a high molecular mass polymer that could be degraded by type 3 polysaccharide-specific depolymerase. As in S. pneumoniae, the membrane-bound synthase from E. coli catalyzed a rapid release of enzyme-bound polysaccharide when incubated with either UDP-Glc or UDP-GlcUA alone. The recombinant enzyme expressed in E. coli was capable of releasing all of the polysaccharide from the enzyme, although the chains remained associated with the membrane. The recombinant enzyme was also able to reinitiate polysaccharide synthesis following polymer release by utilizing a lipid primer present in the membranes. At low concentrations of UDP-Glc and UDP-GlcUA (1 microm in the presence of Mg(2+) and 0.2 microm in Mn(2+)), novel glycolipids composed of repeating disaccharides with linkages consistent with type 3 polysaccharide were synthesized. As the concentration of the UDP-sugars was increased, there was a marked transition from glycolipid to polymer formation. At UDP-sugar concentrations of either 5 microm (with Mg(2+)) or 1.5 microm (with Mn(2+)), 80% of the incorporated sugar was in polymer form, and the size of the polymer increased dramatically as the concentration of UDP-sugars was increased. These results suggest a cooperative interaction between the UDP-precursor-binding site(s) and the nascent polysaccharide-binding site, resulting in a non-processive addition of sugars at the lower UDP-sugar concentrations and a processive reaction as the substrate concentrations increase.     

Characterization of the Lipid Linkage Region and Chain Length of the Cellubiuronic Acid Capsule of Streptococcus pneumoniae

The processive reaction mechanisms of β-glycosyl-polymerases are poorly understood. The cellubiuronan synthase of Streptococcus pneumoniae catalyzes the synthesis of the type 3 capsular polysaccharide through the alternate additions of β-1,3-Glc and β-1,4-GlcUA. The processive multistep reaction involves the sequential binding of two nucleotide sugar donors in coordination with the extension of a polysaccharide chain associated with the carbohydrate acceptor recognition site. Degradation analysis using cellubiuronan-specific depolymerase demonstrated that the oligosaccharide-lipid and polysaccharide-lipid products synthesized in vitro with recombinant cellubiuronan synthase had a similar oligosaccharyl-lipid at their reducing termini, providing definitive evidence for a precursor-product relationship and also confirming that growth occurred at the nonreducing end following initiation on phosphatidylglycerol. The presence of a lipid marker at the reducing end allowed the quantitative determination of cellubiuronic acid polysaccharide chain lengths. As the UDP-GlcUA concentration was increased from 1 to 11.5 μm, the level of synthase in the transitory processive state decreased, with the predominant oligosaccharide-lipid product containing 3 uronic acid residues, whereas the proportion of synthase in the fully processive state increased and the polysaccharide chain length increased from 320 to 6700 monosaccharide units. In conjunction with other kinetic data, these results suggest that the formation of a complex between a tetrauronosyl oligomer and the carbohydrate acceptor recognition site plays a central role in coordinating the repetitive interaction of the synthase with the nucleotide sugar donors and modulating the chain length of cellubiuronan polysaccharide.Cellubiuronic acid, the capsular polysaccharide of type 3 Streptococcus pneumoniae, is composed of the repeating disaccharide cellobiuronic acid (-3)-β-d-GlcUA²-(1,4)-β-d-Glc-(1-) (1) and is synthesized by a processive mechanism similar to that for cellulose, chitin, hyaluronic acid, and other related β-glycans (2). This group of polysaccharides is synthesized by inverting GT-2A polymerases that are located in the plasma membrane with their active sites on the cytoplasmic face, and following chain initiation, the synthases are thought to be involved in the extrusion of the nascent chains to the external membrane face (3–8). The overall processive elaboration of these polysaccharides remains poorly understood at the molecular level. In particular, there is relatively little information concerning the initiation process, the facilitation of chain extrusion, the mechanism of translocation, and the regulation of the final chain length during the assembly of these polymers. Recent investigations in this laboratory have begun to unravel some of the details of both the early and later stages of the biosynthesis of cellubiuronan.Unlike most S. pneumoniae capsules, whose elaboration requires multiple glycosyltransferases, a polymerase, and an additional transport system (9), the assembly and transport of cellubiuronic acid in type 3 strains is carried out by the single enzyme cellubiuronan synthase (Cps3S) (3, 10, 11). Studies of the synthase in S. pneumoniae and recombinant Escherichia coli membranes have shown that assembly of the polysaccharide involves two distinct kinetic phases: 1) a transitory processive state wherein the chain is thought to be initiated by the formation of an oligosaccharide-lipid that is loosely associated with the synthase, and 2) a fully processive state in which the polysaccharide is tightly bound to the carbohydrate substrate recognition site, except for a brief period during the translocation stage of each catalytic cycle (5, 12). Each catalytic cycle in the extrusion mode provides for chain extension by the addition of a repeating disaccharide and requires the alternate association of the synthase with UDP-Glc and UDP-GlcUA, the formation of the glycosidic linkages of the respective sugars, and the release, translocation, and reattachment of the elongating chain at the synthase carbohydrate recognition site. Transition from the transitory mode to the fully processive extrusion mode correlates with the attainment of a threshold-length oligosaccharide of ∼8 sugars (12). Nod factor chito-oligosaccharides from rhizobia are synthesized by a related group of synthases that apparently are not capable of organizing into an extrusion mode (13). Significantly, the maximum length of any reported Nod-factor oligosaccharide is 6 sugars (14).Based on β-glucosidase sensitivity of singly added [¹⁴C]Glc to the terminal end of high molecular weight cellubiuronan, it was deduced that the polysaccharide grows by repetitive β-1,3-Glc and β-1,4-GlcUA additions to the nonreducing terminus (2). Oligosaccharide-lipid assembly is thought to initiate on phosphatidylglycerol (15). To date, however, there has been no quantitative demonstration of polysaccharide-lipid conjugate, and the similarity of the distal sugar-lipid linkages in the polysaccharide- and oligosaccharide-lipid products has not been verified.Cellubiuronan depolymerase is a Bacillus circulans β-endoglucuronidase that specifically cleaves cellubiuronic acid chains at GlcUA-β1,4-Glc linkages (16, 17). The polysaccharide is successively cleaved at random internal linkages, which upon completion of hydrolysis yields a series of oligosaccharide end products containing 1–4 Glc-β1,3-GlcUA disaccharide units, with the most abundant oligomer being a tetrasaccharide. The high degree of specificity of this depolymerase has provided a sensitive analytical tool to further characterize cellubiuronan oligosaccharide- and polysaccharide-lipids, and even more importantly, it has provided a means for determining the chain length of these polysaccharides. Both in vivo (18) and in vitro studies (12) indicate that the processivity of cellubiuronan synthase is modulated by the concentration of UDP-GlcUA. However, lacking well defined cellubiuronan polysaccharide size standards, there has been no way to clearly establish the actual length of the polymer synthesized under different reaction conditions. The methodology described in this article has allowed a clear demonstration of the relationship between the polysaccharide size and the UDP-GlcUA substrate concentration and in turn has led to the development of a kinetic model (38), which provides both for UDP-sugar modulation of polysaccharide chain length and for the assembly by a single-site synthase of a polysaccharide composed of a repeating heterodisaccharide.     

Biosynthesis of type 3 capsular polysaccharide in Streptococcus pneumoniae. Enzymatic chain release by an abortive translocation process

The type 3 polysaccharide synthase from Streptococcus pneumoniae catalyzes sugar transfer from UDP-Glc and UDP-glucuronic acid (GlcUA) to a polymer with the repeating disaccharide unit of [3)-beta-d-GlcUA-(1-->4)-beta-d-Glc-(1-->]. Evidence is presented that release of the polysaccharide chains from S. pneumoniae membranes is time-, temperature-, and pH-dependent and saturable with respect to specific catalytic metabolites. In these studies, the membrane-bound synthase was shown to catalyze a rapid release of enzyme-bound polysaccharide when either UDP-Glc or UDP-GlcUA alone was present in the reaction. Only a slow release of polysaccharide occurred when both UDP sugars were present or when both UDP sugars were absent. Chain size was not a specific determinant in polymer release. The release reaction was saturable with increasing concentrations of UDP-Glc or UDP-GlcUA, with respective apparent K(m) values of 880 and 0.004 micrometer. The apparent V(max) was 48-fold greater with UDP-Glc compared with UDP-GlcUA. The UDP-Glc-actuated reaction was inhibited by UDP-GlcUA with an approximate K(i) of 2 micrometer, and UDP-GlcUA-actuated release was inhibited by UDP-Glc with an approximate K(i) of 5 micrometer. In conjunction with kinetic data regarding the polymerization reaction, these data indicate that UDP-Glc and UDP-GlcUA bind to the same synthase sites in both the biosynthetic reaction and the chain release reaction and that polymer release is catalyzed when one binding site is filled and the concentration of the conjugate UDP-precursor is insufficient to fill the other binding site. The approximate energy of activation values of the biosynthetic and release reactions indicate that release of the polysaccharide occurs by an abortive translocation process. These results are the first to demonstrate a specific enzymatic mechanism for the termination and release of a polysaccharide.     

Control of capsular polysaccharide chain length by UDP-sugar substrate concentrations in Streptococcus pneumoniae

Regulation of chain length is essential to the proper functioning of prokaryotic and eukaryotic polysaccharides. Modulation of polymer size by substrate concentration is an attractive but unexplored control mechanism that has been suggested for many polysaccharides. The Streptococcus pneumoniae capsular polysaccharide is essential for virulence, and regulation of its size is critical for survival in different host environments. Synthesis of the type 3 capsule [-4)-beta-d-Glc-(1-3)-beta-d-GlcUA-(1-] from UDP-glucose (UDP-Glc) and UDP-glucuronic acid (UDP-GlcUA) is catalysed by the type 3 synthase, a processive beta-glycosyltransferase, and requires a UDP-Glc dehydrogenase for conversion of UDP-Glc to UDP-GlcUA. Strains containing mutant UDP-Glc dehydrogenases exhibited reduced levels of UDP-GlcUA, along with reductions in total capsule amount and polymer chain length. In both the parent and mutant strains, UDP-Glc levels far exceeded UDP-GlcUA levels, which were very low to undetectable in the absence of blocking synthase activity. The in vivo observations were consistent with in vitro conditions that effect chain termination and ejection of the polysaccharide from the synthase when one substrate is limiting. These data are the first to demonstrate modulation of polysaccharide chain length by substrate concentration and to enable a model for the underlying mechanism. Further, they may have implications for the control of chain length in both prokaryotic and eukaryotic polymers synthesized by similar mechanisms.     

Single-molecule analysis reveals three phases of DNA degradation by an exonuclease

λ exonuclease degrades one strand of duplex DNA in the 5'-to-3' direction to generate a 3' overhang required for recombination. Its ability to hydrolyze thousands of nucleotides processively is attributed to its ring structure, and most studies have focused on the processive phase. Here we have used single-molecule fluorescence resonance energy transfer (FRET) to reveal three phases of λ exonuclease reactions: the initiation, distributive and processive phases. The distributive phase comprises early reactions in which the 3' overhang is too short to stably engage with the enzyme. A mismatched base is digested one-fifth as quickly as a Watson-Crick-paired base, and multiple concatenated mismatches have a cooperatively negative effect, highlighting the crucial role of base pairing in aligning the 5' end toward the active site. The rate-limiting step during processive degradation seems to be the post-cleavage melting of the terminal base pair. We also found that an escape from a known pausing sequence requires enzyme backtracking.     

Structure and mechanism of human UDP-glucose 6-dehydrogenase

Elevated production of the matrix glycosaminoglycan hyaluronan is strongly implicated in epithelial tumor progression. Inhibition of synthesis of the hyaluronan precursor UDP-glucuronic acid (UDP-GlcUA) therefore presents an emerging target for cancer therapy. Human UDP-glucose 6-dehydrogenase (hUGDH) catalyzes, in two NAD(+)-dependent steps without release of intermediate aldehyde, the biosynthetic oxidation of UDP-glucose (UDP-Glc) to UDP-GlcUA. Here, we present a structural characterization of the hUGDH reaction coordinate using crystal structures of the apoenzyme and ternary complexes of the enzyme bound with UDP-Glc/NADH and UDP-GlcUA/NAD(+). The quaternary structure of hUGDH is a disc-shaped trimer of homodimers whose subunits consist of two discrete α/β domains with the active site located in the interdomain cleft. Ternary complex formation is accompanied by rigid-body and restrained movement of the N-terminal NAD(+) binding domain, sequestering substrate and coenzyme in their reactive positions through interdomain closure. By alternating between conformations in and out of the active site during domain motion, Tyr(14), Glu(161), and Glu(165) participate in control of coenzyme binding and release during 2-fold oxidation. The proposed mechanism of hUGDH involves formation and breakdown of thiohemiacetal and thioester intermediates whereby Cys(276) functions as the catalytic nucleophile. Stopped-flow kinetic data capture the essential deprotonation of Cys(276) in the course of the first oxidation step, allowing the thiolate side chain to act as a trap of the incipient aldehyde. Because thiohemiacetal intermediate accumulates at steady state under physiological reaction conditions, hUGDH inhibition might best explore ligand binding to the NAD(+) binding domain.     

A processive carbohydrate polymerase that mediates bifunctional catalysis using a single active site

Even in the absence of a template, glycosyltransferases can catalyze the synthesis of carbohydrate polymers of specific sequence. The paradigm has been that one enzyme catalyzes the formation of one type of glycosidic linkage, yet certain glycosyltransferases generate polysaccharide sequences composed of two distinct linkage types. In principle, bifunctional glycosyltransferases can possess separate active sites for each catalytic activity or one active site with dual activities. We encountered the fundamental question of one or two distinct active sites in our investigation of the galactosyltransferase GlfT2. GlfT2 catalyzes the formation of mycobacterial galactan, a critical cell-wall polymer composed of galactofuranose residues connected with alternating, regioisomeric linkages. We found that GlfT2 mediates galactan polymerization using only one active site that manifests dual regioselectivity. Structural modeling of the bifunctional glycosyltransferases hyaluronan synthase and cellulose synthase suggests that these enzymes also generate multiple glycosidic linkages using a single active site. These results highlight the versatility of glycosyltransferases for generating polysaccharides of specific sequence. We postulate that a hallmark of processive elongation of a carbohydrate polymer by a bifunctional enzyme is that one active site can give rise to two separate types of glycosidic bonds.     

Identification of active site residues in glucosylceramide synthase. A nucleotide-binding catalytic motif conserved with processive beta-glycosyltransferases

Glucosylceramide synthase (GCS) transfers glucose from UDP-Glc to ceramide, catalyzing the first glycosylation step in the formation of higher order glycosphingolipids. The amino acid sequence of GCS was reported to be dissimilar from other proteins, with no identifiable functional domains. We previously identified His-193 of rat GCS as an important residue in UDP-Glc and GCS inhibitor binding; however, little else is known about the GCS active site. Here, we identify key residues of the GCS active site by performing biochemical and site-directed mutagenesis studies of rat GCS expressed in bacteria. First, we found that Cys-207 was the primary residue involved in GCS N-ethylmaleimide sensitivity. Next, we showed by multiple alignment that the region of GCS flanking His-193 and Cys-207 (amino acids 89-278) contains a D1,D2,D3,(Q/R)XXRW motif found in the putative active site of processive beta-glycosyltransferases (e.g. cellulose, chitin, and hyaluronan synthases). Site-directed mutagenesis studies demonstrated that most of the highly conserved residues were essential for GCS activity. We also note that GCS and processive beta-glycosyltransferases are topologically similar, possessing cytosolic active sites, with putative transmembrane domains immediately N-terminal to the conserved domain. These results provide the first extensive information on the GCS active site and show that GCS and processive beta-glycosyltransferases possess a conserved substrate-binding/catalytic domain.     

Time-resolved charge translocation by the Ca-ATPase from sarcoplasmic reticulum after an ATP concentration jump

Time-resolved measurements of currents generated by Ca-ATPase from fragmented sarcoplasmic reticulum (SR) are described. SR vesicles spontaneously adsorb to a black lipid membrane acting as a capacitive electrode. Charge translocation by the enzyme is initiated by an ATP concentration jump performed by the light-induced conversion of an inactive precursor (caged ATP) to ATP with a time constant of 2.0 ms at pH 6.2 and 24 degrees C. The shape of the current signal is triphasic, an initial current flow into the vesicle lumen is followed by an outward current and a second slow inward current. The time course of the current signal can be described by five relaxation rate constants, lambda1 to lambda5 plus a fixed delay D approximately 1-3 ms. The electrical signal shows that 1) the reaction cycle of the Ca-ATPase contains two electrogenic steps; 2) positive charge is moved toward the luminal side in the first rapid step and toward the cytoplasmic side in the second slow step; 3) at least one electroneutral reaction precedes the electrogenic steps. Relaxation rate constant lambda3 reflects ATP binding, with lambda(3,max) approximately 100 s(-1). This step is electroneutral. Comparison with the kinetics of the reaction cycle shows that the first electrogenic step (inward current) occurs before the decay of E2P. Candidates are the formation of phosphoenzyme from E1ATP (lambda2 approximately 200 s[-1]) and the E1P --> E2P transition (D approximately 1 ms or lambda1 approximately 300 s[-1]). The second electrogenic transition (outward current) follows the formation of E2P (lambda4 approximately 3 s[-1]) and is tentatively assigned to H+ countertransport after the dissociation of Ca2+. Quenched flow experiments performed under the conditions of the electrical measurements 1) demonstrate competition by caged ATP for ATP-dependent phosphoenzyme formation and 2) yield a rate constant for phosphoenzyme formation of 200 s(-1). These results indicate that ATP and caged ATP compete for the substrate binding site, as suggested by the ATP dependence of lambda3 and favor correlation of lambda2 with phosphoenzyme formation.     

The N-terminal carbohydrate recognition site of the cation-independent mannose 6-phosphate receptor

The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR) plays a critical role in the trafficking of newly synthesized mannose 6-phosphate-containing acid hydrolases to the lysosome. The receptor contains two high affinity carbohydrate recognition sites within its 15-domain extracytoplasmic region, with essential residues for carbohydrate recognition located in domain 3 and domain 9. Previous studies have shown that these two sites are distinct with respect to carbohydrate specificity. In addition, expression of truncated forms of the CI-MPR demonstrated that domain 9 can be expressed as an isolated domain, retaining high affinity (Kd approximately 1 nm) carbohydrate binding, whereas expression of domain 3 alone resulted in a protein capable of only low affinity binding (Kd approximately 1 microm) toward a lysosomal enzyme. In the current report the crystal structure of the N-terminal 432 residues of the CI-MPR, encompassing domains 1-3, was solved in the presence of bound mannose 6-phosphate. The structure reveals the unique architecture of this carbohydrate binding pocket and provides insight into the ability of this site to recognize a variety of mannose-containing sugars.     

DNA contacts stimulate catalysis by a poxvirus topoisomerase

Eukaryotic type 1B topoisomerases act by forming covalent enzyme-DNA intermediates that transiently nick DNA and thereby release DNA supercoils. Here we present a study of the topoisomerase encoded by the pathogenic poxvirus molluscum contagiosum. Our studies of DNA sites favored for catalysis reveal a larger recognition site than the 5'-(T/C)CCTT-3' sequence previously identified for poxvirus topoisomerases. Separate assays of initial DNA binding and covalent complex formation revealed that different DNA sequences were important for each reaction step. The location of the protein-DNA contacts was mapped by analyzing mutant sites and inosine-substituted DNAs. Some of the bases flanking the 5'-(T/C)CCTT-3' sequence were selectively important for covalent complex formation but not initial DNA binding. Interactions important for catalysis were probed with 5'-bridging phosphorothiolates at the site of strand cleavage, which permitted covalent complex formation but prevented subsequent religation. Kinetic studies revealed that the flanking sequences that promoted recovery of covalent complexes increased initial cleavage instead of inhibiting resealing of the nicked intermediate. These data 1) indicate that previously unidentified DNA contacts can accelerate a step between initial binding and covalent complex formation and 2) help specify models for conformational changes promoting catalysis.     

Molecular basis of the activity of the phytopathogen pectin methylesterase

We provide a mechanism for the activity of pectin methylesterase (PME), the enzyme that catalyses the essential first step in bacterial invasion of plant tissues. The complexes formed in the crystal using specifically methylated pectins, together with kinetic measurements of directed mutants, provide clear insights at atomic resolution into the specificity and the processive action of the Erwinia chrysanthemi enzyme. Product complexes provide additional snapshots along the reaction coordinate. We previously revealed that PME is a novel aspartic-esterase possessing parallel beta-helix architecture and now show that the two conserved aspartates are the nucleophile and general acid-base in the mechanism, respectively. Other conserved residues at the catalytic centre are shown to be essential for substrate binding or transition state stabilisation. The preferential binding of methylated sugar residues upstream of the catalytic site, and demethylated residues downstream, drives the enzyme along the pectin molecule and accounts for the sequential pattern of demethylation produced by both bacterial and plant PMEs.     

Purification and properties of mouse thymus thymidylate synthase. Comparison of the enzyme from mammalian normal and tumour tissues

Mouse thymus thymidylate synthase has been purified to apparent electrophoretic homogeneity and compared with the enzyme from mouse tumour L1210 and Ehrlich ascites carcinoma cells. The enzyme is a dimer composed of 35,000 mol. wt monomers. Mouse thymus and tumour enzymes exhibit allosteric properties reflected by cooperative binding of both dUMP and 5-fluoro-dUMP. Activation energy for the reaction, catalyzed by thymidylate synthase from mouse tumour but not from mouse thymus, lowers at temperatures above 34 degrees C, reflecting a change of rate-limiting step in dTMP formation. MgATP at millimolar concentrations inhibits mouse thymus enzyme.     

Mechanism of type 3 capsular polysaccharide synthesis in Streptococcus pneumoniae

The glycosidic linkages of the type 3 capsular polysaccharide of Streptococcus pneumoniae ([3)-beta-D-GlcUA-(1-->4)-beta-D-Glc-(1-->](n)) are formed by the membrane-associated type 3 synthase (Cps3S), which is capable of synthesizing polymer from UDP sugar precursors. Using membrane preparations of S. pneumoniae in an in vitro assay, we observed type 3 synthase activity in the presence of either Mn(2+) or Mg(2+) with maximal levels seen with 10-20 mM Mn(2+). High molecular weight polymer synthesized in the assay was composed of Glc and glucuronic acid and could be degraded to a low molecular weight product by a type 3-specific depolymerase from Bacillus circulans. Additionally, the polymer bound specifically to an affinity column made with a type 3 polysaccharide-specific monoclonal antibody. The polysaccharide was rapidly synthesized from smaller chains and remained associated with the enzyme-containing membrane fraction throughout its synthesis, indicating a processive mechanism of synthesis. Release of the polysaccharide was observed, however, when the level of one of the substrates became limiting. Finally, addition of sugars to the growing type 3 polysaccharide was shown to occur at the nonreducing end of the polysaccharide chain.     

Stopped-flow kinetic analysis of the ligand-induced coil-helix transition in glutathione S-transferase A1-1: evidence for a persistent denatured state.

Structural studies have suggested that the glutathione S-transferase (GST) A1-1 isozyme contains a dynamic C-terminus which undergoes a ligand-dependent disorder-order transition and sequesters substrates within the active site. Here, the contribution of the C-terminus to the kinetics and thermodynamics of ligand binding and dissociation has been determined. Steady-state turnover rates of the wild type (WT) and a C-terminal truncated (Delta209-222) rGST A1-1 with ethacrynic acid (EA) were measured in the presence of variable concentrations of viscogen. The results indicate that a physical step involving segmental protein motion is at least partially rate limiting at temperatures between 10 and 40 degrees C for WT. Dissociation rates of the glutathione-ethacrynic acid product conjugate (GS-EA), determined by stopped-flow fluorescence, correspond to the steady-state turnover rates. In contrast, the chemical step governs the turnover reaction by Delta209-222, suggesting that the slow rate of product release for WT is controlled by the dynamics of the C-terminal coil-helix transition. In addition, the association reaction of WT rGST A1-1 with GS-EA established that the binding was biphasic and included ligand docking followed by slow isomerization of the enzyme-ligand complex. In contrast, binding of GS-EA to Delta209-222 was a monophasic, bimolecular reaction. These results indicate that the binding of GS-EA to WT rGST A1-1 proceeds via an induced fit mechanism, with a slow conformational step that corresponds to the coil-helix transition. However, the biphasic dissociation kinetics for the wild type, and the recovered kinetic parameters, suggest that a significant fraction of the [GST.GS-EA] complex ( approximately 15%) retains a persistent disordered state at equilibrium.     

Abietadiene synthase catalysis: conserved residues involved in protonation-initiated cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate

Abietadiene synthase catalyzes two sequential, mechanistically distinct cyclization reactions in the formation of a mixture of abietadiene double bond isomers as the committed step in resin acid biosynthesis. Each reaction is carried out at a separate active site residing in a structurally distinct domain, and the reactions are kinetically separable. The first cyclization reaction is initiated by protonation of the terminal double bond of the universal diterpene precursor, geranylgeranyl diphosphate. The pH dependence of the overall reaction is consistent with an acid-base catalytic mechanism, and a divalent metal ion plays a role in this reaction probably by binding the diphosphate moiety to assist in positioning the substrate for catalysis. A putative active site for the protonation-initiated cyclization was defined by modeling abietadiene synthase and locating the DXDD motif previously shown to be involved in this reaction. A number of charged and aromatic residues, which are highly conserved in mechanistically related diterpene cyclases, line the putative active site. Alanine substitutions were made for each of these residues, as were asparagine and glutamate substitutions for the aspartates of the DXDD motif. Kinetic evaluation confirmed the involvement of most of the targeted residues in the reaction, and analysis of mutational effects on the pH-activity profile and affinity for a transition state analogue suggested specific roles for several of these residues in catalyzing the cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate. A functional role was also suggested for the cryptic insertional element found in abietadiene synthase and other diterpene synthases that carry out similar protonation-initiated cyclizations.     

Relationship between the self-splicing activity and the solidity of the master domain of the Tetrahymena group I ribozyme

The highly conserved P3-P7 domain of the Group I intron ribozymes is known to contain essential elements, such as the binding site for the cofactor guanosine, required for conducting the splicing reaction. We investigated the domain of the Tetrahymena intron ribozyme and its variants in order to clarify the relationship between its stability and function. We found that the destabilization of the P3-P7 domain facilitates the active structure formation at high magnesium ion concentrations where the formation is retarded for the wild type. The destabilized domain also increases K(GTP)(m) although this can be compensated by increasing the concentration of Mg(2+), indicating that the stable domain is required for establishing a tight guanosine binding site. The results suggest that the stability of the domain affects the rate-limiting step in the RNA folding pathway and also regulates the efficiency of the splicing reaction.     

Phase separations of alpha-tocopherol in aqueous dispersions of distearoylphosphatidylethanolamine

The effect of alpha-tocopherol on the structure and thermotropic phase behaviour of distearoylphosphatidylethanolamine was examined by using synchrotron X-ray diffraction methods. There was evidence that alpha-tocopherol does not distribute randomly in the dispersed phospholipid but instead phospholipid phases enriched in alpha-tocopherol are formed. Heating codispersions from lamellar gel phase induced formation of hexagonal-II phase at temperatures below the main transition of the pure phospholipid and which were enriched in alpha-tocopherol. Codispersions containing 5 or 10 mol% alpha-tocopherol were induced to form a cubic phase at temperatures above the lamellar to hexagonal-II phase transition. Such phases were not observed in codispersions containing 2.5 or 20 mol% alpha-tocopherol in which only lamellar and hexagonal-II phases were formed. The space group of the cubic phases were tentatively assigned as Pn3m. Equilibration of codispersions at 4 degrees C results in the formation of lamellar crystalline phases enriched in alpha-tocopherol and phase separated domains of pure phospholipid. Two lamellar crystalline phases were characterized on the basis of their particular wide-angle X-ray scattering patterns. The lamellar crystalline phases were also distinguished from other lamellar phases of the pure phospholipid by the lamellar repeat. Partitioning of alpha-tocopherol into phosphatidylethanolamine domains in membranes may introduce instability into the structure.     

The EcoRI restriction endonuclease with bacteriophage lambda DNA. Kinetic studies.

The kinetics of the reactions of the EcoRI restriction endonuclease at individual recognition sites on the DNA from bacteriophage lambda were found to differ markedly from site to site. Under certain conditions of pH and ionic strength, the rates for the cleavage of the DNA were the same at each recognition site. But under altered experimental conditions, different reaction rates were observed at each recognition site. These results are consistent with a mechanism in which the kinetic stability of the complex between the enzyme and the recognition site on the DNA differs among the sites, due to the effect of interactions between the enzyme and DNA sequences surrounding each recognition site upon the transition state of the reaction. Reactions at individual sites on a DNA molecule containing more than one recognition site were found to be independent of each other, thus excluding the possibility of a processive mechanism for the EcoRI enzyme. The consequences of these observations are discussed with regard to both DNA-protein interactions and to the application of restriction enzymes in the study of the structure of DNA molecules.