Lipases provide a new mechanistic model for polyhydroxybutyrate (PHB) synthases: characterization of the functional residues in Chromatium vinosum PHB synthase

Polyhydroxybutyrate (PHB) synthases catalyze the conversion of beta-hydroxybutyryl coenzyme A (HBCoA) to PHB. These enzymes require an active site cysteine nucleophile for covalent catalysis. A protein BLASTp search using the Class III Chromatium vinosum synthase sequence reveals high homology to prokaryotic lipases whose crystal structures are known. The homology is very convincing in the alpha-beta-elbow (with the active site nucleophile)-alpha-beta structure, residues 131-175 of the synthase. A conserved histidine of the Class III PHB synthases aligns with the active site histidine of the lipases using the ClustalW algorithm. This is intriguing as this histidine is approximately 200 amino acids removed in sequence space from the catalytic nucleophile. Different threading algorithms suggest that the Class III synthases belong to the alpha/beta hydrolase superfamily which includes prokaryotic lipases. Mutagenesis studies were carried out on C. vinosum synthase C149, H331, H303, D302, and C130 residues. These studies reveal that H331 is the general base catalyst that activates the nucleophile, C149, for covalent catalysis. The model indicates that C130 is not involved in catalysis as previously proposed [Müh, U., Sinskey, A. J., Kirby, D. P., Lane, W. S., and Stubbe, J. (1999) Biochemistry 38, 826-837]. Studies with D302 mutants suggest D302 functions as a general base catalyst in activation of the 3-hydroxyl of HBCoA (or a hydroxybutyrate acyl enzyme) for nucleophilic attack on the covalently linked thiol ester intermediate. The relationship of the lipase model to previous models based on fatty acid synthases is discussed.     

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.     

Characterization of a transient covalent adduct formed during dimethylarginine dimethylaminohydrolase catalysis

Dimethylarginine dimethylaminohydrolase (DDAH) regulates the concentrations of human endogenous inhibitors of nitric oxide synthase, N(omega)-methyl-l-arginine (NMMA), and asymmetric N(omega),N(omega)-dimethyl-l-arginine (ADMA). Pharmacological regulation of nitric oxide synthesis is an important goal, but the catalytic mechanism of DDAH remains largely unexplored. A DDAH from Pseudomonas aeruginosa was cloned, and asymmetrically methylated arginine analogues were shown to be the preferred substrates, with ADMA displaying a slightly higher k(cat)/K(M) value than NMMA. DDAH is similar to members of a larger superfamily of guanidino-modifying enzymes, some of which have been shown to use an S-alkylthiouronium intermediate during catalysis. No covalent intermediates were found to accumulate during steady-state turnover reactions of DDAH with NMMA or ADMA. However, identification of a new substrate with an activated leaving group, S-methyl-l-thiocitrulline (SMTC), enabled acid trapping and ESI-MS characterization of a transient covalent adduct with a mass of 158 +/- 10 Da that accumulates during steady-state turnover. Subsequent trapping, proteolysis, peptide mapping and fragmentation by mass spectrometry, and site-directed mutagenesis demonstrated that this covalent adduct was attached to an active site residue and implicates Cys249 as the catalytic nucleophile required for intermediate formation. The use of covalent catalysis clearly links DDAH to this superfamily of enzymes and suggests that an S-alkylthiouronium intermediate may be a conserved feature in their mechanisms.     

The role of glutamic acid-69 in the activation of Citrobacter freundii tyrosine phenol-lyase by monovalent cations

Tyrosine phenol-lyase (TPL) from Citrobacter freundii is activated about 30-fold by monovalent cations, the most effective being K(+), NH(4)(+), and Rb(+). Previous X-ray crystal structure analysis has demonstrated that the monovalent cation binding site is located at the interface between subunits, with ligands contributed by the carbonyl oxygens of Gly52 and Asn262 from one chain and monodentate ligation by one of the epsilon-oxygens of Glu69 from another chain [Antson, A. A., Demidkina, T. V., Gollnick, P., Dauter, Z., Von Tersch, R. L., Long, J., Berezhnoy, S. N., Phillips, R. S., Harutyunyan, E. H., and Wilson, K. S. (1993) Biochemistry 32, 4195]. We have studied the effect of mutation of Glu69 to glutamine (E69Q) and aspartate (E69D) to determine the role of Glu69 in the activation of TPL. E69Q TPL is activated by K(+), NH(4)(+), and Rb(+), with K(D) values similar to wild-type TPL, indicating that the negative charge on Glu69 is not necessary for cation binding and activation. In contrast, E69D TPL exhibits very low basal activity and only weak activation by monovalent cations, even though monovalent cations are capable of binding, indicating that the geometry of the monovalent cation binding site is critical for activation. Rapid-scanning stopped-flow kinetic studies of wild-type TPL show that the activating effect of the cation is seen in an acceleration of rates of quinonoid intermediate formation (30-50-fold) and of phenol elimination. Similar rapid-scanning stopped-flow results were obtained with E69Q TPL; however, E69D TPL shows only a 4-fold increase in the rate of quinonoid intermediate formation with K(+). Preincubation of TPL with monovalent cations is necessary to observe the rate acceleration in stopped flow kinetic experiments, suggesting that the activation of TPL by monovalent cations is a slow process. In agreement with this conclusion, a slow increase (k < 0.5 s(-)(1)) in fluorescence intensity (lambda(ex) = 420 nm, lambda(em) = 505 nm) is observed when wild-type and E69Q TPL are mixed with K(+), Rb(+), and NH(4)(+) but not Li(+) or Na(+). E69D TPL shows no change in fluorescence under these conditions. High concentrations (>100 mM) of all monovalent cations result in inhibition of wild-type TPL. This inhibition is probably due to cation binding to the ES complex to form a complex that releases pyruvate slowly.     

The Catalytic Mechanism of the Hotdog-fold Enzyme Superfamily 4-Hydroxybenzoyl-CoA Thioesterase from Arthrobacter sp. Strain SU

The hotdog-fold enzyme 4-hydroxybenzoyl-coenzyme A (4-HB-CoA) thioesterase from Arthrobacter sp. strain AU catalyzes the hydrolysis of 4-HB-CoA to form 4-hydroxybenzoate (4-HB) and coenzyme A (CoA) in the final step of the 4-chlorobenzoate dehalogenation pathway. Guided by the published X-ray structures of the liganded enzyme (Thoden, J. B., Zhuang, Z., Dunaway-Mariano, D., and Holden H. M. (2003) J.Biol. Chem. 278, 43709-43716), a series of site-directed mutants were prepared for testing the roles of active site residues in substrate binding and catalysis. The mutant thioesterases were subjected to X-ray structure determination to confirm retention of the native fold, and in some cases, to reveal changes in the active site configuration. In parallel, the wild-type and mutant thioesterases were subjected to transient and steady-state kinetic analysis, and to (18)O-solvent labeling experiments. Evidence is provided that suggests that Glu73 functions in nucleophilic catalysis, that Gly65 and Gln58 contribute to transition-state stabilization via hydrogen bond formation with the thioester moiety and that Thr77 orients the water nucleophile for attack at the 4-hydroxybenzoyl carbon of the enzyme-anhydride intermediate. The replacement of Glu73 with Asp was shown to switch the function of the carboxylate residue from nucleophilic catalysis to base catalysis and thus, the reaction from a two-step process involving a covalent enzyme intermediate to a single-step hydrolysis reaction. The E73D/T77A double mutant regained most of the catalytic efficiency lost in the E73D single mutant. The results from (31)P NMR experiments indicate that the substrate nucleotide unit is bound to the enzyme surface. Kinetic analysis of site-directed mutants was carried out to determine the contributions made by Arg102, Arg150, Ser120, and Thr121 in binding the nucleotide unit. Lastly, we show by kinetic and X-ray analyses of Asp31, His64, and Glu78 site-directed mutants that these three active site residues are important for productive binding of the substrate 4-hydroxybenzoyl ring.     

A familiar motif in a new context: the catalytic mechanism of hydroxyisourate hydrolase

Hydroxyisourate hydrolase is a recently discovered enzyme that participates in the ureide pathway in soybeans. Its role is to catalyze the hydrolysis of 5-hydroxyisourate, the product of the urate oxidase reaction. There is extensive sequence homology between hydroxyisourate hydrolase and retaining glycosidases; in particular, the conserved active site glutamate residues found in retaining glycosidases are present in hydroxyisourate hydrolase as Glu 199 and Glu 408. However, experimental investigation of their roles, as well as the catalytic mechanism of the enzyme, have been precluded by the instability of 5-hydroxyisourate. Here, we report that diaminouracil serves as a slow, alternative substrate and can be used to investigate catalysis by hydroxyisourate hydrolase. The activity of the E199A protein was reduced 400-fold relative to wild-type, and no activity could be detected with the E408A mutant. Steady-state kinetic studies of the wild-type protein revealed that the pH-dependence of V(max) and V/K describe bell-shaped curves, consistent with the hypothesis that catalysis requires two ionizable groups in opposite protonation states. Addition of 100 mM azide accelerated the reaction catalyzed by the wild-type enzyme 8-fold and the E199A mutant 20-fold but had no effect on the E408A mutant. These data suggest that Glu 408 acts as a nucleophile toward the substrate forming a covalent anhydride intermediate, and Glu 199 facilitates formation of the intermediate by serving as a general acid and then activates water for hydrolysis of the intermediate. Thus, the mechanism of hydroxyisourate hydrolase is strikingly similar to that of retaining glycosidases, even though it catalyzes hydrolysis of an amide bond.     

Stereoelectronic control of bond formation in Escherichia coli tryptophan synthase: substrate specificity and enzymatic synthesis of the novel amino acid dihydroisotryptophan

The reactions of the indole analogues indoline and aniline with the Escherichia coli tryptophan synthase alpha-aminoacrylate Schiff base intermediate have been characterized by UV-visible and 1H NMR absorption spectroscopy and compared with the interactions of indole and the potent inhibitor benzimidazole. Indole, via the enamine functionality of the pyrrole ring, reacts with the alpha-aminoacrylate intermediate, forming a transient quinonoid species with lambda max 476 nm as the new C-C bond is synthesized. Conversion of this quinonoid to L-tryptophan is the rate-limiting step in catalysis [Lane, A., & Kirschner, K. (1981) Eur. J. Biochem. 120, 379-398]. Both aniline and indoline undergo rapid N-C bond formation with the alpha-aminoacrylate to form quinonoid intermediates; benzimidazole binds rapidly and tightly to the alpha-aminoacrylate but does not undergo covalent bond formation. The indoline and aniline quinonoids (lambda max 464 and 466 nm, respectively) are formed via nucleophilic attack on the electrophilic C-beta of the alpha-aminoacrylate. The indoline quinonoid decays slowly, yielding a novel, new amino acid, dihydroisotryptophan. The aniline quinonoid is quasi-stable, and no new amino acid product was detected. We conclude that nucleophilic attack requires the precise alignment of bonding orbitals between nucleophile and the alpha-aminoacrylate intermediate. The constraints imposed by the geometry of the indole subsite force the aromatic rings of indoline, aniline, and benzimidazole to bind in the same plane as indole; thus nucleophilic attack occurs with the N-1 atoms of indoline and aniline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stabilization of HIV-1 envelope in the CD4-bound conformation through specific cross-linking of a CD4 mimetic

CD4 binding on gp120 leads to the exposure of highly conserved regions recognized by the HIV co-receptor CCR5 and by CD4-induced (CD4i) antibodies. A covalent gp120-CD4 complex was shown to elicit CD4i antibody responses in monkeys, which was correlated with control of the HIV virus infection (DeVico, A., Fouts, T., Lewis, G. K., Gallo, R. C., Godfrey, K., Charurat, M., Harris, I., Galmin, L., and Pal, R. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 17477-17482). Because the inclusion of CD4 in a vaccine formulation should be avoided, due to potential autoimmune reactions, we engineered small sized CD4 mimetics (miniCD4s) that are poorly immunogenic and do not induce anti-CD4 antibodies. We made covalent complexes between such an engineered miniCD4 and gp120 or gp140, through a site-directed coupling reaction. These complexes were recognized by CD4i antibodies as well as by the HIV co-receptor CCR5. In addition, they elicit CD4i antibody responses in rabbits and therefore represent potential vaccine candidates that mimic an important HIV fusion intermediate, without autoimmune hazard.     

Glutamic acid 274 is the nucleophile in the active site of a "retaining" exoglucanase from Cellulomonas fimi.

In addition to its known substrate activity with p-nitrophenyl beta-cellobioside, the exoglucanase from Cellulomonas fimi has been shown to utilize substituted phenyl beta-glucosides as substrates, of which the best is 2',4'-dinitrophenyl beta-D-glucopyranoside. The enzyme can be inactivated by treatment with 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-glucopyranoside, by trapping of the covalent intermediate in catalysis, as has been shown for a beta-glucosidase (Withers, S.G., and Street, I.P. (1988) J. Am. Chem. Soc. 110, 8551-8553). The intermediate formed is stable but can undergo turnover in the presence of cellobiose, reactivating the enzyme by transglycosylation. Using a tritium-labeled inactivator it has been possible to isolate and sequence a radiolabeled peptide from this enzyme, and the active site nucleophile has been identified as glutamic acid residue 274. This glutamic acid residue and its sequentially proximal amino acids are absolutely conserved in the homologous family F of cellulases.     

Mechanism-based inhibitors of CD38: a mammalian cyclic ADP-ribose synthetase

The soluble domain of human CD38 catalyzes the conversion of NAD(+) to cyclic ADP-ribose and to ADP-ribose via a common covalent intermediate [Sauve, A. A., Deng, H. T., Angelletti, R. H., and Schramm, V. L. (2000) J. Am. Chem. Soc. 122, 7855-7859]. Here we establish that mechanism-based inhibitors can be produced by chemical stabilization of this intermediate. The compounds nicotinamide 2'-deoxyriboside (1), 5-methylnicotinamide 2'-deoxyriboside (2), and pyridyl 2'-deoxyriboside (3) were synthesized and evaluated as inhibitors for human CD38. The nicotinamide derivatives 1 and 2 were inhibitors of the enzyme as determined by competitive behavior in CD38-catalyzed conversion of nicotinamide guanine dinucleotide (NGD(+)) to cyclic GDP-ribose. The K(i) values for competitive inhibition were 1.2 and 4.0 microM for 1 and 2, respectively. Slow-onset characteristics of reaction progress curves indicated a second higher affinity state of these two inhibitors. Inhibitor off-rates were slow with rate constants k(off) of 1.5 x 10(-5) s(-1) for 1 and 2.5 x 10(-5) s(-1) for 2. Apparent dissociation constants K(i(total)) for 1 and 2 were calculated to be 4.5 and 12.5 nM, respectively. The similar values for k(off) are consistent with the hydrolysis of common enzymatic intermediates formed by the reaction of 1 and 2 with the enzyme. Both form covalently attached deoxyribose groups to the catalytic site nucleophile. Chemical evidence for this intermediate is the ability of nicotinamide to rescue enzyme activity after inactivation by either 1 or 2. A covalent intermediate is also indicated by the ability of CD38 to catalyze base exchange, as observed by conversion of 2 to 1 in the presence of nicotinamide. The deoxynucleosides 1 and 2 demonstrate that the chemical determinants for mechanism-based inhibition of CD38 can be satisfied by nucleosides that lack the 5'-phosphate, the adenylate group, and the 2'-hydroxyl moiety. In addition, these compounds reveal the mechanism of CD38 catalysis to proceed by the formation of a covalent intermediate during normal catalytic turnover with faster substrates. The covalent 2'-deoxynucleoside inactivators of CD38 are powerful inhibitors by acting as good substrates for formation of the covalent intermediate but are poor leaving groups from the intermediate complex because hydrolytic assistance of the 2'-hydroxyl group is lacking. The removal of the adenylate nucleophile required for the cyclization reaction provides slow hydrolysis as the only exit from the covalent complex.     

Substrate-induced DNA Polymerase β Activation

DNA polymerases and substrates undergo conformational changes upon forming protein-ligand complexes. These conformational adjustments can hasten or deter DNA synthesis and influence substrate discrimination. From structural comparison of binary DNA and ternary DNA-dNTP complexes of DNA polymerase β, several side chains have been implicated in facilitating formation of an active ternary complex poised for chemistry. Site-directed mutagenesis of these highly conserved residues (Asp-192, Arg-258, Phe-272, Glu-295, and Tyr-296) and kinetic characterization provides insight into the role these residues play during correct and incorrect insertion as well as their role in conformational activation. The catalytic efficiencies for correct nucleotide insertion for alanine mutants were wild type ∼ R258A > F272A ∼ Y296A > E295A > D192A. Because the efficiencies for incorrect insertion were affected to about the same extent for each mutant, the effects on fidelity were modest (<5-fold). The R258A mutant exhibited an increase in the single-turnover rate of correct nucleotide insertion. This suggests that the wild-type Arg-258 side chain generates a population of non-productive ternary complexes. Structures of binary and ternary substrate complexes of the R258A mutant and a mutant associated with gastric carcinomas, E295K, provide molecular insight into intermediate structural conformations not appreciated previously. Although the R258A mutant crystal structures were similar to wild-type enzyme, the open ternary complex structure of E295K indicates that Arg-258 stabilizes a non-productive conformation of the primer terminus that would decrease catalysis. Significantly, the open E295K ternary complex binds two metal ions indicating that metal binding cannot overcome the modified interactions that have interrupted the closure of the N-subdomain.     

Site-directed mutagenesis of glutamate 317 of bovine alpha-1,3Galactosyltransferase and its effect on enzyme activity: implications for reaction mechanism

Bovine alpha1,3galactosyltransferase (alpha1,3GalT) transfers galactose from UDP-alpha-galactose to terminal beta-linked galactosyl residues with retention of configuration of the incoming galactose residue. The epitope synthesized has been shown to be critical for xenotransplantation. According to a proposed double-displacement reaction mechanism, glutamate-317 (E317) is thought to be the catalytic nucleophile. The proposed catalytic role of E317 involves an initial nucleophilic attack with inversion of configuration and formation of a covalent sugar-enzyme intermediate between E317 and galactose from the donor substrate, followed by a second nucleophilic attack performed by the acceptor substrate with a second inversion of configuration. To determine whether E317 of alpha1,3GalT is critical for enzyme activity, site-directed mutagenesis was used to substitute alanine, aspartic acid, cysteine and histidine for E317. If the proposed reaction mechanism for the alpha1,3GalT enzyme is correct, E317D and E317H would produce active enzymes since they can act as nucleophiles. The non-conservative mutation E317A and conservative mutation E317C are predicted to produce inactive or very low activity enzymes since the E317A mutant cannot engage in a nucleophilic attack, and the E317C mutant would trap the galactose residue. The results obtained demonstrate that E317D and E317H mutants retained activity, albeit significantly less than the wild-type enzyme. Additionally, both E317A and E317C mutant also retained enzyme activity, suggesting that E317 is not the catalytic nucleophile proposed in the double-displacement mechanism. Therefore, either a different amino acid may act as the catalytic nucleophile or the reaction must proceed by a different mechanism.     

Reaction mechanism of pyridoxal 5'-phosphate synthase. Detection of an enzyme-bound chromophoric intermediate

Vitamin B6 is an essential metabolite in all organisms. De novo synthesis of the vitamin can occur through either of two mutually exclusive pathways referred to as deoxyxylulose 5-phosphate-dependent and deoxyxylulose 5-phosphate-independent. The latter pathway has only recently been discovered and is distinguished by the presence of two genes, Pdx1 and Pdx2, encoding the synthase and glutaminase subunit of PLP synthase, respectively. In the presence of ammonia, the synthase alone displays an exceptional polymorphic synthetic ability in carrying out a complex set of reactions, including pentose and triose isomerization, imine formation, ammonia addition, aldol-type condensation, cyclization, and aromatization, that convert C3 and C5 precursors into the cofactor B6 vitamer, pyridoxal 5'-phosphate. Here, employing the Bacillus subtilis proteins, we demonstrate key features along the catalytic path. We show that ribose 5-phosphate is the preferred C5 substrate and provide unequivocal evidence that the pent(ul)ose phosphate imine occurs at lysine 81 rather than lysine 149 as previously postulated. While this study was under review, corroborative crystallographic evidence has been provided for imine formation with the corresponding lysine group in the enzyme from Thermotoga maritima (Zein, F., Zhang, Y., Kang, Y.-N., Burns, K., Begley, T. P., and Ealick, S. E. (2006) Biochemistry 45, 14609-14620). We have detected an unanticipated covalent reaction intermediate that occurs subsequent to imine formation and is dependent on the presence of Pdx2 and glutamine. This step most likely primes the enzyme for acceptance of the triose sugar, ultimately leading to formation of the pyridine ring. Two alternative structures are proposed for the chromophoric intermediate, both of which require substantial modifications of the proposed mechanism.     

3-Hydroxy-3-methylglutaryl-CoA synthase: participation of invariant acidic residues in formation of the acetyl-S-enzyme reaction intermediate

Inactivation of HMG-CoA synthase by a carboxyl-directed reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), in a concentration-dependent and substrate-protectable manner suggested that the active site contains reactive acidic amino acids. This observation prompted functional evaluation of 11 invariant acidic amino acids by site-directed mutagenesis. Characterization of the isolated synthase variants' ability to catalyze overall and partial reactions identified three mutant synthases (D99A, D159A, and D203A) that exhibit significant diminution of k(cat) for the overall reaction (10(2)-, 10(3)-, and 10(4)-fold decreases, respectively). D99A, D159A, and D203A form the acetyl-S-enzyme intermediate very slowly (0.0025, 0.0026, 0.0015 U/mg, respectively, measured at pH 7. 0 and 22 degrees C) as compared to the wild-type synthase (1.59 U/mg), where intermediate formation approaches rate-limiting status. Differences in substrate saturation do not account for impaired activities or rates of intermediate formation. The structural integrity of the purified mutants' active sites is demonstrated by their abilities to bind a spin-labeled acyl-CoA analogue (R.CoA) with affinities and stoichiometries comparable to values measured for wild-type synthase. The impact of three distinct amino acids on reaction intermediate formation supports a mechanism of acetyl-S-enzyme formation that probably requires formation and directed collapse of a tetrahedral adduct. (18)O-induced shift of the (13)C NMR signal of (13)C acetyl-S-enzyme demonstrates that an analogous tetrahedral species is produced upon solvent exchange with the acetyl-S-enzyme. Partial discrimination between the functions of D99, D159, and D203 becomes possible based on the observation that D159A and D203A synthases exhibit retarded kinetics of solvent (18)O exchange while D99A fails to support (18)O exchange.     

Utility of acetyldithio-CoA in detecting the influence of active site residues on substrate enolization by 3-hydroxyl-3-methylglutaryl-CoA synthase

Hydroxymethylglutaryl-CoA synthase-catalyzed condensation of acetyl-CoA with acetoacetyl-CoA requires enolization/carbanion formation from the acetyl C-2 methyl group prior to formation of a new carbon-carbon bond. Acetyldithio-CoA, a readily enolizable analog of acetyl-CoA, was an effective competitive inhibitor of avian hydroxymethylglutaryl-CoA synthase (Ki = 28 microm). In the absence of cosubstrate, enzyme catalyzed the enolization/proton exchange from the C-2 methyl group of acetyldithio-CoA. Mutant enzymes that exhibited impaired formation of the covalent acetyl-S-enzyme reaction intermediate exhibited diminished (D159A and D203A) or undetectable (C129S) rates of enolization of acetyldithio-CoA. The results suggest that covalent thioacetylation of protein, which has not been detected previously for other enzymes that enolize this analog, occurs with hydroxymethylglutaryl-CoA synthase. Enzyme catalyzed the transfer of the thioacetyl group of this analog to 3'-dephospho-CoA suggesting the intermediacy of a covalent thioacetyl-S-enzyme species, which appears to be important for proton abstraction from C-2 of the thioacetyl group. Avian enzyme glutamate 95 is crucial to substrate condensation to form a new carboncarbon bond. Mutations of this invariant residue (avian enzyme E95A and E95Q; Staphylococcus aureus enzyme E79Q) correlated with diminished ability to catalyze enolization of acetyldithio-CoA. Enolization by E95Q was not stimulated in the presence of acetoacetyl-CoA. These observations suggest either a direct (proton abstraction) or indirect (solvent polarization) role for this active site glutamate.     

Reaction mechanism of glutathione synthetase from Arabidopsis thaliana: site-directed mutagenesis of active site residues

Glutathione is essential for maintaining the intracellular redox environment and is synthesized from gamma-glutamylcysteine, glycine, and ATP by glutathione synthetase (GS). To examine the reaction mechanism of a eukaryotic GS, 24 Arabidopsis thaliana GS (AtGS) mutants were kinetically characterized. Within the gamma-glutamylcysteine/glutathione-binding site, the S153A and S155A mutants displayed less than 4-fold changes in kinetic parameters with mutations of Glu-220 (E220A/E220Q), Gln-226 (Q226A/Q226N), and Arg-274 (R274A/R274K) at the distal end of the binding site resulting in 24-180-fold increases in the K(m) values for gamma-glutamylcysteine. Substitution of multiple residues interacting with ATP (K313M, K367M, and E429A/E429Q) or coordinating magnesium ions to ATP (E148A/E148Q, N150A/N150D, and E371A) yielded inactive protein because of compromised nucleotide binding, as determined by fluorescence titration. Other mutations in the ATP-binding site (E371Q, N376A, and K456M) resulted in greater than 30-fold decreases in affinity for ATP and up to 80-fold reductions in turnover rate. Mutation of Arg-132 and Arg-454, which are positioned at the interface of the two substrate-binding sites, affected the enzymatic activity differently. The R132A mutant was inactive, and the R132K mutant decreased k(cat) by 200-fold; however, both mutants bound ATP with K(d) values similar to wild-type enzyme. Minimal changes in kinetic parameters were observed with the R454K mutant, but the R454A mutant displayed a 160-fold decrease in k(cat). In addition, the R132K, R454A, and R454K mutations elevated the K(m) value for glycine up to 11-fold. Comparison of the pH profiles and the solvent deuterium isotope effects of A. thaliana GS and the Arg-132 and Arg-454 mutants also suggest distinct mechanistic roles for these residues. Based on these results, a catalytic mechanism for the eukaryotic GS is proposed.     

Loop Residues and Catalysis in OMP Synthase

Residue-to-alanine mutations and a two-amino acid deletion have been made in the highly conserved catalytic loop (residues 100-109) of Salmonella typhimurium OMP synthase (orotate phosphoribosyltransferase, EC 2.4.2.10). As described previously, the K103A mutant enzyme exhibited a 10(4)-fold decrease in k(cat)/K(M) for PRPP; the K100A enzyme suffered a 50-fold decrease. Alanine mutations at His105 and Glu107 produced 40- and 7-fold decreases in k(cat)/K(M), respectively, and E101A, D104A, and G106A were slightly faster than the wild-type (WT) in terms of k(cat), with minor effects on k(cat)/K(M). Equilibrium binding of OMP or PRPP in binary complexes was affected little by loop mutation, suggesting that the energetics of ground-state binding have little contribution from the catalytic loop, or that a favorable binding energy is offset by costs of loop reorganization. Pre-steady-state kinetics for mutants showed that K103A and E107A had lost the burst of product formation in each direction that indicated rapid on-enzyme chemistry for WT, but that the burst was retained by H105A. Δ102Δ106, a loop-shortened enzyme with Ala102 and Gly106 deleted, showed a 10(4)-fold reduction of k(cat) but almost unaltered K(D) values for all four substrate molecules. The 20% (i.e., 1.20) intrinsic [1'-(3)H]OMP kinetic isotope effect (KIE) for WT is masked because of high forward and reverse commitment factors. K103A failed to express intrinsic KIEs fully (1.095 ± 0.013). In contrast, H105A, which has a smaller catalytic lesion, gave a [1'-(3)H]OMP KIE of 1.21 ± 0.0005, and E107A (1.179 ± 0.0049) also gave high values. These results are interpreted in the context of the X-ray structure of the complete substrate complex for the enzyme [Grubmeyer, C., Hansen, M. R., Fedorov, A. A., and Almo, S. C. (2012) Biochemistry 51 (preceding paper in this issue, DOI 10.1021/bi300083p )]. The full expression of KIEs by H105A and E107A may result from a less secure closure of the catalytic loop. The lower level of expression of the KIE by K103A suggests that in these mutant proteins the major barrier to catalysis is successful closure of the catalytic loop, which when closed, produces rapid and reversible catalysis.     

The P450cam G248E mutant covalently binds its prosthetic heme group

Previous studies on mammalian peroxidases and cytochrome P450 family 4 enzymes have shown that a carboxylic group positioned close to a methyl group of the prosthetic heme is required for the formation of a covalent link between a protein carboxylic acid side chain and the heme. To determine whether there are additional requirements for covalent bond formation in the P450 enzymes, a glutamic acid or an aspartic acid has been introduced into P450(cam) close to the heme 5-methyl group. Spectroscopic and kinetic studies of the resulting G248E and G248D mutants suggest that the carboxylate group coordinates with the heme iron atom, as reported for a comparable P450(BM3) mutant [Girvan, H. M., Marshall, K. R., Lawson, R. J., Leys, D., Joyce, M. G., Clarkson, J., Smith, W. E., Cheesman, M. R., and Munro, A. W. (2004) J. Biol. Chem. 279, 23274-23286]. The two P450(cam) mutants have low catalytic activity, but in contrast to the P450(BM3) mutant, incubation of the G248E (but not G248D) mutant with camphor, putidaredoxin, putidaredoxin reductase, and NADH results in partial covalent binding of the heme to the protein. No covalent attachment is observed in the absence of camphor or any of the other reaction components. Pronase digestion of the G248E P450(cam) mutant after covalent attachment of the heme releases 5-hydroxyheme, establishing that the heme is covalently attached through its 5-methyl group as predicted by in silico modeling. The results establish that a properly positioned carboxyl group is the sole requirement for autocatalytic formation of a heme-protein link in P450 enzymes, but also show that efficient covalent binding requires placement of the carboxyl close to the methyl but in a manner that prevents strong coordination to the iron atom.     

Analysis of a catalytic pathway via a covalent adduct of D52E hen egg white mutant lysozyme by further mutation.

We previously demonstrated by X-ray crystallography and electrospray mass spectrometry that D52E mutant hen lysozyme formed a covalent enzyme-substrate adduct on reaction with N-acetylglucosamine oligomer. This observation indicates that D52E lysozyme may acquire a catalytic pathway via a covalent adduct. To explain this pathway, the formation and hydrolysis reactions of the covalent adduct were investigated. Kinetic analysis indicated that the hydrolysis step was the rate-limiting step, 60-fold slower than the formation reaction. In the formation reaction, the pH dependence was bell-shaped, which was plausibly explained by the functions of the two catalytic pKas of Glu35 and Glu52. On the other hand, the pH dependence in the hydrolysis was sigmoidal with a transition at pH 4. 5, which was identical with the experimentally determined pKa of Glu35 in the covalent adduct, indicating that Glu35 functions as a general base to hydrolyze the adduct. To improve the turnover rate of D52E lysozyme, the mutation of N46D was designed and introduced to D52E lysozyme. This mutation reduced the activation energy in the hydrolysis reaction of the covalent adduct by 1.8 kcal/mol at pH 5.0 and 40 degrees C but did not affect the formation reaction. Our data may provide a useful approach to understanding the precise mechanism of the function of natural glycosidases, which catalyze via a covalent adduct.