Mechanism of the addition half of the O-acetylserine sulfhydrylase-A reaction

O-Acetylserine sulfhydrylase (OASS) catalyzes the last step in the cysteine biosynthetic pathway in enteric bacteria and plants, substitution of the beta-acetoxy group of O-acetyl-l-serine (OAS) with inorganic bisulfide. The first half of the sulfhydrylase reaction, formation of the alpha-aminoacrylate intermediate, limits the overall reaction rate, while in the second half-reaction, with bisulfide as the substrate, chemistry is thought to be diffusion-limited. In order to characterize the second half-reaction, the pH dependence of the pseudo-first-order rate constant for disappearance of the alpha-aminoacrylate intermediate was measured over the pH range 6.0-9.5 using the natural substrate bisulfide, and a number of nucleophilic analogues. The rate is pH-dependent for substrates with a pK(a) > 7, while the rate constant is pH-independent for substrates with a pK(a) < 7 suggesting that the pK(a)s of the substrate and an enzyme group are important in this half of the reaction. In D(2)O, at low pD values, the amino acid external Schiff base is trapped, while in H(2)O the reaction proceeds through release of the amino acid product, which is likely rate-limiting for all nucleophilic reactants. A number of new beta-substituted amino acids were produced and characterized by (1)H NMR spectroscopy.     

Crystal structure of a sulfur carrier protein complex found in the cysteine biosynthetic pathway of Mycobacterium tuberculosis

The structure of the protein complex CysM-CysO from a new cysteine biosynthetic pathway found in the H37Rv strain of Mycobacterium tuberculosis has been determined at 1.53 A resolution. CysM (Rv1336) is a PLP-containing beta-replacement enzyme and CysO (Rv1335) is a sulfur carrier protein with a ubiquitin-like fold. CysM catalyzes the replacement of the acetyl group of O-acetylserine by CysO thiocarboxylate to generate a protein-bound cysteine that is released in a subsequent proteolysis reaction. The protein complex in the crystal structure is asymmetric with one CysO protomer binding to one end of a CysM dimer. Additionally, the structures of CysM and CysO were determined individually at 2.8 and 2.7 A resolution, respectively. Sequence alignments with homologues and structural comparisons with CysK, a cysteine synthase that does not utilize a sulfur carrier protein, revealed high conservation of active site residues; however, residues in CysM responsible for CysO binding are not conserved. Comparison of the CysM-CysO binding interface with other sulfur carrier protein complexes revealed a similarity in secondary structural elements that contribute to complex formation in the ThiF-ThiS and MoeB-MoaD systems, despite major differences in overall folds. Comparison of CysM with and without bound CysO revealed conformational changes associated with CysO binding.     

The C-terminal of CysM from Mycobacterium tuberculosis protects the aminoacrylate intermediate and is involved in sulfur donor selectivity

A new crystal structure of the dimeric cysteine synthase CysM from Mycobacterium tuberculosis reveals an open and a closed conformation of the enzyme. In the closed conformation the five carboxy-terminal amino acid residues are inserted into the active site cleft. Removal of this segment results in a decreased lifetime of the α-aminoacrylate reaction intermediate, an increased sensitivity to oxidants such as hydrogen peroxide, and loss of substrate selectivity with respect to the sulfur carrier thiocarboxylated CysO. These results highlight features of CysM that might be of particular importance for cysteine biosynthesis under oxidative stress in M. tuberculosis.     

Biotinylation, a post-translational modification controlled by the rate of protein-protein association

Biotin protein ligases catalyze specific covalent linkage of the coenzyme biotin to biotin-dependent carboxylases. The reaction proceeds in two steps, including synthesis of an adenylated intermediate followed by biotin transfer to the carboxylase substrate. In this work specificity in the transfer reaction was investigated using single turnover stopped-flow and quench-flow assays. Cognate and noncognate reactions were measured using the enzymes and minimal biotin acceptor substrates from Escherichia coli, Pyrococcus horikoshii, and Homo sapiens. The kinetic analysis demonstrates that for all enzyme-substrate pairs the bimolecular rate of association of enzyme with substrate limits post-translational biotinylation. In addition, in noncognate reactions the three enzymes displayed a range of selectivities. These results highlight the importance of protein-protein binding kinetics for specific biotin addition to carboxylases and provide one mechanism for determining biotin distribution in metabolism.     

Aminoacrylate intermediates in the reaction of Citrobacter freundii tyrosine phenol-lyase

Tyrosine phenol-lyase (TPL) from Citrobacter freundii is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the reversible hydrolytic cleavage of l-Tyr to give phenol and ammonium pyruvate. The proposed reaction mechanism for TPL involves formation of an external aldimine of the substrate, followed by deprotonation of the alpha-carbon to give a quinonoid intermediate. Elimination of phenol then has been proposed to give an alpha-aminoacrylate Schiff base, which releases iminopyruvate that ultimately undergoes hydrolysis to yield ammonium pyruvate. Previous stopped-flow kinetic experiments have provided direct spectroscopic evidence for the formation of the external aldimine and quinonoid intermediates in the reactions of substrates and inhibitors; however, the predicted alpha-aminoacrylate intermediate has not been previously observed. We have found that 4-hydroxypyridine, a non-nucleophilic analogue of phenol, selectively binds and stabilizes aminoacrylate intermediates in reactions of TPL with S-alkyl-l-cysteines, l-tyrosine, and 3-fluoro-l-tyrosine. In the presence of 4-hydroxypyridine, a new absorption band at 338 nm, assigned to the alpha-aminoacrylate, is observed with these substrates. Formation of the 338 nm peaks is concomitant with the decay of the quinonoid intermediates, with good isosbestic points at approximately 365 nm. The value of the rate constant for aminoacrylate formation is similar to k(cat), suggesting that leaving group elimination is at least partially rate limiting in TPL reactions. In the reaction of S-ethyl-l-cysteine in the presence of 4-hydroxypyridine, a subsequent slow reaction of the alpha-aminoacrylate is observed, which may be due to iminopyruvate formation. Both l-tyrosine and 3-fluoro-l-tyrosine exhibit kinetic isotope effects of approximately 2-3 on alpha-aminoacrylate formation when the alpha-(2)H-labeled substrates are used, consistent with the previously reported internal return of the alpha-proton to the phenol product. These results are the first direct spectroscopic observation of alpha-aminoacrylate intermediates in the reactions of TPL.     

Structure and mechanism of O-acetylserine sulfhydrylase

The O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium catalyzes a beta-replacement reaction in which the beta-acetoxy group of O-acetyl-L-serine (OAS) is replaced by bisulfide to give L-cysteine and acetate. The kinetic mechanism of OASS is ping-pong with a stable alpha-aminoacrylate intermediate. The enzyme is a homodimer with one pyridoxal 5'-phosphate (PLP) bound per subunit deep within the protein in a cleft between the N- and C-terminal domains of each of the monomers. All of the active site residues are contributed by a single subunit. The enzyme cycles through open and closed conformations as it catalyzes its reaction with structural changes largely limited to a subdomain of the N-terminal domain. The elimination of acetic acid from OAS is thought to proceed via an anti-E2 mechanism, and the only catalytic group identified to date is lysine 41, which originally participates in Schiff base linkage to PLP. The transition state for the elimination of acetic acid is thought to be asynchronous and earlier for Cbeta-O bond cleavage than for Calpha-H bond cleavage.     

The three transglycosylation reactions catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans (strain 251) proceed via different kinetic mechanisms.

Cyclodextrin glycosyltransferase (CGTase) catalyzes three transglycosylation reactions via a double displacement mechanism involving a covalent enzyme-intermediate complex (substituted-enzyme intermediate). Characterization of the three transglycosylation reactions, however, revealed that they differ in their kinetic mechanisms. Disproportionation (cleavage of an alpha-glycosidic bond of a linear malto-oligosaccharide and transfer of one part to an acceptor substrate) proceeds according to a ping-pong mechanism. Cyclization (cleavage of an alpha-glycosidic bond in amylose or starch and subsequent formation of a cyclodextrin) is a single-substrate reaction with an affinity for the high molecular mass substrate used, which was too high to allow elucidation of the kinetic mechanism. Michaelis-Menten kinetics, however, have been observed using shorter amylose chains. Coupling (cleavage of an alpha-glycosidic bond in a cyclodextrin ring and transfer of the resulting linear malto-oligosaccharide to an acceptor substrate) proceeds according to a random ternary complex mechanism. In view of the different kinetic mechanisms observed for the various reactions, which can be related to differences in substrate binding, it should be possible to mutagenize CGTase in such a manner that a single reaction is affected most strongly. Construction of CGTase mutants that synthesize linear oligosaccharides instead of cyclodextrins thus appears feasible. Furthermore, the rate of interconversion of linear and circular conformations of oligosaccharides in the cyclization and coupling reactions was found to determine the reaction rate. In the cyclization reaction this conversion rate, together with initial binding of the high molecular mass substrate, may determine the product specificity of the enzyme. These new insights will allow rational design of CGTase mutant enzymes synthesizing cyclodextrins of specific sizes.     

Substitution of glutamic acid 109 by aspartic acid alters the substrate specificity and catalytic activity of the beta-subunit in the tryptophan synthase bienzyme complex from Salmonella typhimurium.

In an effort to understand the catalytic mechanism of the tryptophan synthase beta-subunit from Salmonella typhimurium, possible functional active site residues have been identified (on the basis of the 3-D crystal structure of the bienzyme complex) and targeted for analysis utilizing site-directed mutagenesis. The chromophoric properties of the pyridoxal 5'-phosphate cofactor provide a particularly convenient and sensitive spectral probe to directly investigate changes in catalytic events which occur upon modification of the beta-subunit. Substitution of Asp for Glu 109 in the beta-subunit was found to alter both the catalytic activity and the substrate specificity of the beta-reaction. Steady-state kinetic data reveal that the beta-reaction catalyzed by the beta E109D alpha 2 beta 2 mutant enzyme complex is reduced 27-fold compared to the wild-type enzyme. Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy shows that the mutation does not seriously affect the pre-steady-state reaction of the beta E109D mutant with L-serine to form the alpha-aminoacrylate intermediate, E(A-A). Binding of the alpha-subunit specific ligand, alpha-glycerol phosphate (GP) to the alpha 2 beta 2 complex exerts the same allosteric effects on the beta-subunit as observed with the wild-type enzyme. However, the pre-steady-state spectral changes for the reaction of indole with E(A-A) show that the formation of the L-tryptophan quinonoid, E(Q3), is drastically altered. Discrimination against E(Q3) formation is also observed for the binding of L-tryptophan to the mutant alpha 2 beta 2 complex in the reverse reaction. In contrast, substitution of Asp for Glu 109 increases the apparent affinity of the beta E109D alpha-aminoacrylate complex for the indole analogue indoline and results in the increased rate of synthesis of the amino acid product dihydroiso-L-tryptophan. Thus, the mutation affects the covalent bond forming addition reactions and the nucleophile specificity of the beta-reaction catalyzed by the bienzyme complex.     

Beta D305A mutant of tryptophan synthase shows strongly perturbed allosteric regulation and substrate specificity.

Substrate channeling in the tryptophan synthase bienzyme is regulated by allosteric interactions. Allosteric signals are transmitted via a scaffolding of structural elements that includes a monovalent cation-binding site and salt-bridging interactions between the side chains of betaAsp 305, betaArg 141, betaLys 167, and alphaAsp 56 that appear to modulate the interconversion between open and closed conformations. betaAsp 305 also interacts with the hydroxyl group of the substrate L-Ser in some structures. One possible functional role for betaAsp 305 is to ensure the allosteric transmission that triggers the switching of alphabeta-dimeric units between open and closed conformations of low and high activity. This work shows that substitution of betaAsp 305 with Ala (betaD305A) decreases the affinity of the beta-site for the substrate L-Ser, destabilizes the enzyme-bound alpha-aminoacrylate, E(A-A), and quinonoid species, E(Q), and changes the nucleophile specificity of the beta-reaction. The altered specificity provides a biosynthetic route for new L-amino acids derived from substrate analogues. betaD305A also shows an increased rate of formation of pyruvate upon reaction with L-Ser relative to the wild-type enzyme. The formation of pyruvate is strongly inhibited by the binding of benzimidazole to E(A-A). Upon reaction with L-Ser and in the presence of the alpha-site substrate analogue, alpha-glycerol phosphate, the Na(+) form of betaD305A undergoes inactivation via reaction of nascent alpha-aminoacrylate with bound PLP. This work establishes important roles for betaAsp 305 both in the conformational change between open and closed states that takes place at the beta-site during the formation of the E(A-A) and in substrate binding and recognition.     

Investigation of the mechanism of phosphinothricin inactivation of Escherichia coli glutamine synthetase using rapid quench kinetic technique.

A number of slow tight-binding inhibitors are known for glutamine synthetase that resemble the geometry of the tetrahedral intermediate formed during the enzyme-catalyzed condensation of gamma-glutamyl phosphate and ammonia. One of these inhibitors, phosphinothricin [L-2-amino-4-(hydroxymethyl-phosphinyl)butanoic acid], has been investigated by rapid kinetic methods. Phosphinothricin not only exhibits the kinetic properties of a slow tight-binding inhibitor but also undergoes phosphorylation during the course of the ATP-dependent inactivation. The acid lability of phosphinothricin phosphate enabled investigation of the kinetics of glutamine synthetase inactivation using rapid quench kinetic techniques. The rate-limiting step in the inhibition reaction is the binding of inhibitor (0.004-0.014 microM-1 s-1) and/or a conformational change associated with binding, which is several orders of magnitude slower than the binding of ATP. The association rate of phosphinothricin depends on which metal ion is bound to the enzyme (Mn2+ or Mg2+). With Mn2+ bound to glutamine synthetase the rate of association and the phosphorylation rate are faster than when Mg2+ is bound. The data are interpreted with use of a model in which the binding of a substrate analogue with a tetrahedral moiety enhances the phosphorylation rate of the reaction intermediate; however, the initial binding interaction is retarded because the enzyme has to bind a molecule that has a "transition-state" geometry rather than a ground-state substrate structure. During the course of the inactivation, progressively slower rates for binding and phosphoryl transfer were observed, indicating communication between active sites.     

Proton transfers in the beta-reaction catalyzed by tryptophan synthase

Reactions catalyzed by the beta-subunits of the tryptophan synthase alpha(2)beta(2) complex involve multiple covalent transformations facilitated by proton transfers between the coenzyme, the reacting substrates, and acid-base catalytic groups of the enzyme. However, the UV/Vis absorbance spectra of covalent intermediates formed between the pyridoxal 5'-phosphate coenzyme (PLP) and the reacting substrate are remarkably pH-independent. Furthermore, the alpha-aminoacrylate Schiff base intermediate, E(A-A), formed between L-Ser and enzyme-bound PLP has an unusual spectrum with lambda(max) = 350 nm and a shoulder extending to greater than 500 nm. Other PLP enzymes that form E(A-A) species exhibit intense bands with lambda(max) approximately 460-470 nm. To further investigate this unusual tryptophan synthase E(A-A) species, these studies examine the kinetics of H(+) release in the reaction of L-Ser with the enzyme using rapid kinetics and the H(+) indicator phenol red in solutions weakly buffered by substrate L-serine. This work establishes that the reaction of L-Ser with tryptophan synthase gives an H(+) release when the external aldimine of L-Ser, E(Aex(1)), is converted to E(A-A). This same H(+) release occurs in the reaction of L-Ser plus the indole analogue, aniline, in a step that is rate-determining for the appearance of E(Q)(Aniline). We propose that the kinetic and spectroscopic properties of the L-Ser reaction with tryptophan synthase reflect a mechanism wherein the kinetically detected proton release arises from conversion of an E(Aex(1)) species protonated at the Schiff base nitrogen to an E(A-A) species with a neutral Schiff base nitrogen. The mechanistic and conformational implications of this transformation are discussed.     

Catalytic importance of the substrate binding order for the FMNH2-dependent alkanesulfonate monooxygenase enzyme

The two-component alkanesulfonate monooxygenase system from Escherichia coli includes an FMN reductase (SsuE) and an FMNH2-dependent alkanesulfonate monooxygenase (SsuD) involved in the acquisition of sulfur from alkanesulfonates during sulfur starvation. The SsuD enzyme directly catalyzes the oxidation of alkanesulfonate to aldehyde and sulfite in the presence of O2 and FMNH2. The goal of these studies was to investigate the kinetic mechanism of SsuD through rapid reaction kinetics and substrate binding studies. The SsuD enzyme shows a clear preference for FMNH2 (Kd, 0.32 +/- 0.15 microM) compared to FMN (Kd, 10.2 +/- 0.4 microM) with a 1:1 binding stoichiometry for each form of the flavin. The kinetic trace of premixed SsuD and FMNH2 mixed with oxygenated buffer was best fit to a double exponential with no observed formation of the C4a-(hydro)peroxyflavin. However, when FMNH2 was mixed with SsuD and oxygenated buffer an initial fast phase (kobs, 12.9 s-1) was observed, suggesting that the mixing order is critical for the accumulation of the C4a-(hydro)peroxyflavin. Results from fluorimetric titrations with octanesulfonate imply that reduced flavin must bind first to promote octanesulfonate binding. When octanesulfonate was included in the kinetic studies the C4a-(hydro)peroxyflavin was observed at 370 nm when FMNH2 was not premixed with SsuD, which correlated with an increase in octanal product. There was a clear hyperbolic dependence on octanesulfonate binding, indicating that octanesulfonate binds in rapid equilibrium, and further results indicated there was a second isomerization step following binding. These results suggest that an ordered substrate binding mechanism is important in the desulfonation reaction by SsuD with reduced flavin binding first followed by either O2 or octanesulfonate.     

Cysteine synthase (CysM) of Mycobacterium tuberculosis is an O-phosphoserine sulfhydrylase: evidence for an alternative cysteine biosynthesis pathway in mycobacteria

The biosynthesis of cysteine is a crucial metabolic pathway supplying a building block for de novo protein synthesis but also a reduced thiol as a component of the oxidative defense mechanisms that appear particularly vital in the dormant state of Mycobacterium tuberculosis. We here show that the cysteine synthase CysM is, in contrast to previous annotations, an O-phosphoserine-specific cysteine synthase. CysM belongs to the fold type II pyridoxal 5'-phosphate-dependent enzymes, as revealed by the crystal structure determined at 2.1-angstroms resolution. A model of O-phosphoserine bound to the enzyme suggests a hydrogen bonding interaction of the side chain of Arg220 with the phosphate group as a key feature in substrate selectivity. Replacement of this residue results in a significant loss of specificity for O-phosphoserine. Notably, reactions with sulfur donors are not affected by the amino acid replacement. The specificity of CysM toward O-phosphoserine together with the previously established novel mode of sulfur delivery via thiocarboxylated CysO (Burns, K. E., Baumgart, S., Dorrestein, P. C., Zhai, H., McLafferty, F. W., and Begley, T. P. (2005) J. Am. Chem. Soc. 127, 11602-11603) provide strong evidence for an O-phosphoserine-based cysteine biosynthesis pathway in M. tuberculosis that is independent of both O-acetylserine and the sulfate reduction pathway. The existence of an alternative biosynthetic pathway to cysteine in this pathogen has implications for the design strategy aimed at inhibition of this metabolic route.     

The kinetic mechanism of AAC3-IV aminoglycoside acetyltransferase from Escherichia coli

The aminoglycoside 3-N-acetyltransferase AAC(3)-IV from Escherichia coli exhibits a very broad aminoglycoside specificity, causing resistance to a large number of aminoglycosides, including the atypical veterinary antibiotic, apramycin. We report here on the characterization of the substrate specificity and kinetic mechanism of the acetyl transfer reaction catalyzed by AAC(3)-IV. The steady-state kinetic parameters revealed a narrow specificity for the acyl-donor and broad range of activity for aminoglycosides. AAC(3)-IV has the broadest substrate specificity of all AAC(3)'s studied to date. Dead-end inhibition and ITC experiments revealed that AAC(3)-IV follows a sequential, random bi-bi kinetic mechanism. The analysis of the pH dependence of the kinetic parameters revealed acid- and base-assisted catalysis and the existence of three additional ionizable groups involved in substrate binding. The magnitude of the solvent kinetic isotope effects suggests that a chemical step is at least partially rate limiting in the overall reaction.     

Reaction mechanism of the reconstituted aspartate/glutamate carrier from bovine heart mitochondria

A functional model for the aspartate/glutamate carrier of the inner mitochondrial membrane was established based on a kinetic evaluation of this transporter. Antiport kinetics were measured in proteoliposomes that contained partially purified carrier protein of definite transmembrane orientation (Dierks, T. and Kr?mer, R. (1988) Biochim. Biophys. Acta 937, 122-126). Bireactant initial velocity analyses of the counterexchange reaction were carried out varying substrate concentrations both in the internal and the external compartment. The kinetic patterns obtained were inconsistent with a pong-pong mechanism; rather they demonstrated the formation of a ternary complex as a consequence of sequential binding of one internal and one external substrate molecule to the carrier. Studies on transport activity in the presence of aspartate and glutamate in the same compartment (formally treated as substrate inhibition) clearly indicated that during exchange only one form of the carrier at either membrane surface exposes its binding sites, for which the two different substrates compete. In the deenergized state (pH 6.5) both substrates were translocated at about the same rate. Aspartate/glutamate antiport became asymmetric if a membrane potential was imposed, due to the electrogenic nature of the heteroexchange resulting from proton cotransport together with glutamate. Investigation of the electrical properties of aspartate/aspartate homoexchange led to the conclusion that the translocating carrier-substrate intermediate exhibits a transmembrane symmetry with respect to the (negative) charge, which again only is conceivable assuming a ternary complex. Thus, an antiport model is outlined that shows the functional complex of the carrier with two substrate molecules bound, one at either side of the membrane. The conformational change associated with the transition of both substrate molecules across the membrane then occurs in a single step. Furthermore the model implicates a distinct proton binding site, which is derived from the different influence of H+ concentration observed on transport affinity and transport velocity, respectively, when glutamate is used as a substrate.     

Serine activation is the rate limiting step of tRNASer aminoacylation by yeast seryl tRNA synthetase.

Using the quenched flow technique the mechanism of seryl tRNA synthetase action has been investigated with respect to the presteady state kinetics of individual steps. Under conditions where the strong binding sites of the enzyme are nearly saturated and the steady state turnover number is about 1 s-1, rate constants of four different processes have been determined: steps connected with substrate associations are relatively slow (12 s-1 for the entire process); activation of serine is the rate determining step (about 1.2 s-1 in presence of tRNASer); whereas the transfer of serine onto tRNASer (35 s-1) and the dissociation of seryl tRNASer (70 s-1) are fast. Similar kinetic parameter seem to hold also for the steady state reactions. This conclusion is based on a detailed study of the substrate, product, and Mg2+ concentration dependence of the transfer reaction. The results also indicate that a second serine binding site is operative. Since the transfer of serine from a preformed adenylate complex onto tRNASer is fast, seryl adenylate seems to be a kinetically competent intermediate of the aminoacylation reaction although, of course, alternative mechanisms cannot be excluded.     

Synergistic, random sequential binding of substrates in cobalamin-independent methionine synthase

Cobalamin-independent methionine synthase (MetE) catalyzes the transfer of the N5-methyl group of methyltetrahydrofolate (CH(3)-H(4)folate) to the sulfur of homocysteine (Hcy) to form methionine and tetrahydrofolate (H(4)folate) as products. This reaction is thought to involve a direct methyl transfer from one substrate to the other, requiring the two substrates to interact in a ternary complex. The crystal structure of a MetE.CH(3)-H(4)folate binary complex shows that the methyl group is pointing away from the Hcy binding site and is quite distant from the position where the sulfur of Hcy would be, raising the possibility that this binary complex is nonproductive. The CH(3)-H(4)folate must either rearrange or dissociate before methyl transfer can occur. Therefore, determining the order of substrate binding is of interest. We have used kinetic and equilibrium measurements in addition to isotope trapping experiments to elucidate the kinetic pathway of substrate binding in MetE. These studies demonstrate that both substrate binary complexes are chemically and kinetically competent for methyl transfer and suggest that the conformation observed in the crystal structure is indeed on-pathway. Additionally, the substrates are shown to bind synergistically, with each substrate binding 30-fold more tightly in the presence of the other. Methyl transfer has been determined to be slow compared to ternary complex formation and dissociation. Simulations indicate that nearly all of the enzyme is present as the ternary complex under physiological conditions.     

Functional properties of the active core of human cystathionine beta-synthase crystals

Human cystathionine beta-synthase is a pyridoxal 5'-phosphate enzyme containing a heme binding domain and an S-adenosyl-l-methionine regulatory site. We have investigated by single crystal microspectrophotometry the functional properties of a mutant lacking the S-adenosylmethionine binding domain. Polarized absorption spectra indicate that oxidized and reduced hemes are reversibly formed. Exposure of the reduced form of enzyme crystals to carbon monoxide led to the complete release of the heme moiety. This process, which takes place reversibly and without apparent crystal damage, facilitates the preparation of a heme-free human enzyme. The heme-free enzyme crystals exhibited polarized absorption spectra typical of a pyridoxal 5'-phosphate-dependent protein. The exposure of these crystals to increasing concentrations of the natural substrate l-serine readily led to the formation of the key catalytic intermediate alpha-aminoacrylate. The dissociation constant of l-serine was found to be 6 mm, close to that determined in solution. The amount of the alpha-aminoacrylate Schiff base formed in the presence of l-serine was pH independent between 6 and 9. However, the rate of the disappearance of the alpha-aminoacrylate, likely forming pyruvate and ammonia, was found to increase at pH values higher than 8. Finally, in the presence of homocysteine the alpha-aminoacrylate-enzyme absorption band readily disappears with the concomitant formation of the absorption band of the internal aldimine, indicating that cystathionine beta-synthase crystals catalyze both beta-elimination and beta-replacement reactions. Taken together, these findings demonstrate that the heme moiety is not directly involved in the condensation reaction catalyzed by cystathionine beta-synthase.     

Engineering substrate preference in subtilisin: structural and kinetic analysis of a specificity mutant

Bacillus subtilisin has been a popular model protein for engineering altered substrate specificity. Although some studies have succeeded in increasing the specificity of subtilisin, they also demonstrate that high specificity is difficult to achieve solely by engineering selective substrate binding. In this paper, we analyze the structure and transient state kinetic behavior of Sbt160, a subtilisin engineered to strongly prefer substrates with phenylalanine or tyrosine at the P4 position. As in previous studies, we measure improvements in substrate affinity and overall specificity. Structural analysis of an inactive version of Sbt160 in complex with its cognate substrate reveals improved interactions at the S4 subsite with a P4 tyrosine. Comparison of transient state kinetic behavior against an optimal sequence (DFKAM) and a similar, but suboptimal, sequence (DVRAF) reveals the kinetic and thermodynamic basis for increased specificity, as well as the limitations of this approach. While highly selective substrate binding is achieved in Sbt160, several factors cause sequence specificity to fall short of that observed with natural processing subtilisins. First, for substrate sequences which are nearly optimal, the acylation reaction becomes faster than substrate dissociation. As a result, the level of discrimination among these substrates diminishes due to the coupling between substrate binding and the first chemical step (acylation). Second, although Sbt160 has 24-fold higher substrate affinity for the optimal substrate DFKAM than for DVRAF, the increased substrate binding energy is not translated into improved transition state stabilization of the acylation reaction. Finally, as interactions at subsites become stronger, the rate-determining step in peptide hydrolysis changes from acylation to product release. Thus, the release of the product becomes sluggish and leads to a low k(cat) for the reaction. This also leads to strong product inhibition of substrate turnover as the reaction progresses. The structural and kinetic analysis reveals that differences in the binding modes at subsites for substrates, transition states, and products are subtle and difficult to manipulate via straightforward protein engineering. These findings suggest several new strategies for engineering highly sequence selective enzymes.     

Chemical transformation is not rate-limiting in the reaction catalyzed by Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HMDP). Because HPPK is essential for microorganisms but is absent from human and animals, the enzyme is an excellent target for developing antimicrobial agent. Thermodynamic analysis shows that Mg(2+) is important not only for the binding of nucleotides but also for the binding of HMDP. Transient kinetic analysis shows that a step or steps after the chemical transformation are rate-limiting in the reaction catalyzed by HPPK. The pre-steady-state kinetics is composed of a burst phase and a steady-state phase. The rate constant for the burst phase is approximately 50 times larger than that for the steady-state phase. The latter is very similar to the k(cat) value measured by steady-state kinetics. A set of rate constants for the individual steps of the HPPK-catalyzed reaction has been determined by a combination of stopped-flow and quench-flow analyses. These results form a thermodynamic and kinetic framework for dissecting the roles of active site residues in the substrate binding and catalysis by HPPK.