Ground-state destabilization in orotate phosphoribosyltransferases by binding isotope effects

Orotate phosphoribosyltransferases (OPRTs) form and break the N-ribosidic bond to pyrimidines by way of ribocation-like transition states (TSs) and therefore exhibit large α-secondary 1'-(3)H k(cat)/K(m) kinetic isotope effects (KIEs) [Zhang, Y., and Schramm, V. L. (2010) J. Am. Chem. Soc. 132, 8787-8794]. Substrate binding isotope effects (BIEs) with OPRTs report on the degree of ground-state destabilization for these complexes and permit resolution of binding and transition-state effects from the k(cat)/K(m) KIEs. The BIEs for interactions of [1'-(3)H]orotidine 5'-monophosphate (OMP) with the catalytic sites of Plasmodium falciparum and human OPRTs are 1.104 and 1.108, respectively. These large BIEs establish altered sp(3) bond hybridization of C1' toward the sp(2) geometry of the transition states upon OMP binding. Thus, the complexes of these OPRTs distort OMP part of the way toward the transition state. As the [1'-(3)H]OMP k(cat)/K(m) KIEs are approximately 1.20, half of the intrinsic k(cat)/K(m) KIEs originate from BIEs. Orotidine, a slow substrate for these enzymes, binds to the catalytic site with no significant [1'-(3)H]orotidine BIEs. Thus, OPRTs are unable to initiate ground-state destabilization of orotidine by altered C1' hybridization because of the missing 5'-phosphate. However the k(cat)/K(m) KIEs for [1'-(3)H]orotidine are also approximately 1.20. The C1' distortion for OMP happens in two steps, half upon binding and half on going from the Michaelis complex to the TS. With orotidine as the substrate, there is no ground-state destabilization in the Michaelis complexes, but the C1' distortion at the TS is equal to that of OMP. The large single barrier for TS formation with orotidine slows the rate of barrier crossing.     

Studies of the enzymic mechanism of Candida tenuis xylose reductase (AKR 2B5): X-ray structure and catalytic reaction profile for the H113A mutant

Xylose reductase from the yeast Candida tenuis (CtXR) is a family 2 member of the aldo-keto reductase (AKR) superfamily of proteins and enzymes. Active site His-113 is conserved among AKRs, but a unified mechanism of how it affects catalytic activity is outstanding. We have replaced His-113 by alanine using site-directed mutagenesis, determined a 2.2 A structure of H113A mutant bound to NADP(+), and compared catalytic reaction profiles of NADH-dependent reduction of different aldehydes catalyzed by the wild type and the mutant. Deuterium kinetic isotope effects (KIEs) on k(cat) and k(cat)/K(m xylose) show that, relative to the wild type, the hydride transfer rate constant (k(7) approximately 0.16 s(-1)) has decreased about 1000-fold in H113A whereas xylose binding was not strongly affected. No solvent isotope effect was seen on k(cat) and k(cat)/K(m xylose) for H113A, suggesting that proton transfer has not become rate-limiting as a result of the mutation. The pH profiles of log(k(cat)/K(m xylose)) for the wild type and H113A decreased above apparent pK(a) values of 8.85 and 7.63, respectively. The DeltapK(a) of -1.2 pH units likely reflects a proximally disruptive character of the mutation, affecting the position of Asp-50. A steady-state kinetic analysis for H113A-catalyzed reduction of a homologous series of meta-substituted benzaldehyde derivatives was carried out, and quantitative structure-reactivity correlations were used to factor the observed kinetic substituent effect on k(cat) and k(cat)/K(m aldehyde) into an electronic effect and bonding effects (which are lacking in the wild type). Using the Hammett sigma scale, electronic parameter coefficients (rho) of +0.64 (k(cat)) and +0.78 (k(cat)/K(m aldehyde)) were calculated and clearly differ from rho(k(cat)/K(aldehyde)) and rho(k(cat)) values of +1.67 and approximately 0.0, respectively, for the wild-type enzyme. Hydride transfer rate constants of H113A, calculated from kinetic parameters and KIE data, display a substituent dependence not seen in the corresponding wild-type enzyme rate constants. An enzymic mechanism is proposed in which His-113, through a hydrogen bond from Nepsilon2 to aldehyde O1, assists in catalysis by optimizing the C=O bond charge separation and orbital alignment in the ternary complex.     

Ternary complex structure of human HGPRTase, PRPP, Mg2+, and the inhibitor HPP reveals the involvement of the flexible loop in substrate binding.

Site-directed mutagenesis was used to replace Lys68 of the human hypoxanthine phosphoribosyltransferase (HGPRTase) with alanine to exploit this less reactive form of the enzyme to gain additional insights into the structure activity relationship of HGPRTase. Although this substitution resulted in only a minimal (one- to threefold) increase in the Km values for binding pyrophosphate or phosphoribosylpyrophosphate, the catalytic efficiencies (k(cat)/Km) of the forward and reverse reactions were more severely reduced (6- to 30-fold), and the mutant enzyme showed positive cooperativity in binding of alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) and nucleotide. The K68A form of the human HGPRTase was cocrystallized with 7-hydroxy [4,3-d] pyrazolo pyrimidine (HPP) and Mg PRPP, and the refined structure reported. The PRPP molecule built into the [(Fo - Fc)phi(calc)] electron density shows atomic interactions between the Mg PRPP and enzyme residues in the pyrophosphate binding domain as well as in a long flexible loop (residues Leu101 to Gly111) that closes over the active site. Loop closure reveals the functional roles for the conserved SY dipeptide of the loop as well as the molecular basis for one form of gouty arthritis (S103R). In addition, the closed loop conformation provides structural information relevant to the mechanism of catalysis in human HGPRTase.     

Structure of Salmonella typhimurium OMP Synthase in a Complete Substrate Complex

Dimeric Salmonella typhimurium orotate phosphoribosyltransferase (OMP synthase, EC 2.4.2.10), a key enzyme in de novo pyrimidine nucleotide synthesis, has been cocrystallized in a complete substrate E·MgPRPP·orotate complex and the structure determined to 2.2 ? resolution. This structure resembles that of Saccharomyces cerevisiae OMP synthase in showing a dramatic and asymmetric reorganization around the active site-bound ligands but shares the same basic topology previously observed in complexes of OMP synthase from S. typhimurium and Escherichia coli. The catalytic loop (residues 99-109) contributed by subunit A is reorganized to close the active site situated in subunit B and to sequester it from solvent. Furthermore, the overall structure of subunit B is more compact, because of movements of the amino-terminal hood and elements of the core domain. The catalytic loop of subunit B remains open and disordered, and subunit A retains the more relaxed conformation observed in loop-open S. typhimurium OMP synthase structures. A non-proline cis-peptide formed between Ala71 and Tyr72 is seen in both subunits. The loop-closed catalytic site of subunit B reveals that both the loop and the hood interact directly with the bound pyrophosphate group of PRPP. In contrast to dimagnesium hypoxanthine-guanine phosphoribosyltransferases, OMP synthase contains a single catalytic Mg(2+) in the closed active site. The remaining pyrophosphate charges of PRPP are neutralized by interactions with Arg99A, Lys100B, Lys103A, and His105A. The new structure confirms the importance of loop movement in catalysis by OMP synthase and identifies several additional movements that must be accomplished in each catalytic cycle. A catalytic mechanism based on enzymic and substrate-assisted stabilization of the previously documented oxocarbenium transition state structure is proposed.     

Kinetic and spectroscopic analysis of the catalytic role of H79 in the methionine aminopeptidase from Escherichia coli

To gain insight into the role of the strictly conserved histidine residue, H79, in the reaction mechanism of the methionyl aminopeptidase from Escherichia coli ( EcMetAP-I), the H79A mutated enzyme was prepared. Co(II)-loaded H79A exhibits an overall >7000-fold decrease in specific activity. The almost complete loss of activity is primarily due to a >6000-fold decrease in k cat. Interestingly, the K m value obtained for Co(II)-loaded H79A was approximately half the value observed for wild-type (WT) EcMetAP-I. Consequently, k cat/ K m values decreased only 3000-fold. On the other hand, the observed specific activity of Mn(II)-loaded H79A EcMetAP-I decreased by approximately 2.6-fold while k cat decreased by approximately 3.5-fold. The observed K m value for Mn(II)-loaded H79A EcMetAP-I was approximately 1.4-fold larger than that observed for WT EcMetAP-I, resulting in a k cat/ K m value that is lower by approximately 3.4-fold. Metal binding, UV-vis, and EPR data indicate that the active site is unperturbed by mutation of H79, as suggested by X-ray crystallographic data. Kinetic isotope data indicate that H79 does not transfer a proton to the newly forming amine since a single proton is transferred in the transition state for both the WT and H79A EcMetAP-I enzymes. Therefore, H79 functions to position the substrate by hydrogen bonding to either the amine group of the peptide linkage or a backbone carbonyl group. Together, these data provide new insight into the catalytic mechanism of EcMetAP-I.     

Kinetic and spectroscopic characterization of the H178A methionyl aminopeptidase from Escherichia coli

To gain insight into the role of the strictly conserved histidine residue, H178, in the reaction mechanism of the methionyl aminopeptidase from Escherichia coli (EcMetAP-I), the H178A mutant enzyme was prepared. Metal-reconstituted H178A binds only one equivalent of Co(II) or Fe(II) tightly with affinities that are identical to the WT enzyme based on kinetic and isothermal titration calorimetry (ITC) data. Electronic absorption spectra of Co(II)-loaded H178A EcMetAP-I indicate that the active site divalent metal ion is pentacoordinate, identical to the WT enzyme. These data indicate that the metal binding site has not been affected by altering H178. The effect of altering H178 on activity is, in general, due to a decrease in k(cat). The k(cat) value for Co(II)-loaded H178A decreased 70-fold toward MGMM and 290-fold toward MP-p-NA compared to the WT enzyme, while k(cat) decreased 50-fold toward MGMM for the Fe(II)-loaded H178A enzyme and 140-fold toward MP-p-NA. The K(m) values for MGMM remained unaffected, while those for MP-p-NA increased approximately 2-fold for Co(II)- and Fe(II)-loaded H178A. The k(cat)/K(m) values for both Co(II)- and Fe(II)-loaded H178A toward both substrates ranged from approximately 50- to 580-fold reduction. The pH dependence of log K(m), log k(cat), and log(k(cat)/K(m)) of both WT and H178A EcMetAP-I were also obtained and are identical, within error, for H178A and WT EcMetAP-I. Therefore, H178A is catalytically important but is not required for catalysis. Assignment of one of the observed pK(a) values at 8.1 for WT EcMetAP-I was obtained from plots of molar absorptivity at lambda(max(640)) vs pH for both WT and H178A EcMetAP-I. Apparent pK(a) values of 8.1 and 7.6 were obtained for WT and H178A EcMetAP-I, respectively, and were assigned to the deprotonation of a metal-bound water molecule. The data reported herein provide support for the key elements of the previously proposed mechanism and suggest that a similar mechanism can apply to the enzyme with a single metal in the active site.     

Vibrationally enhanced hydrogen tunneling in the Escherichia coli thymidylate synthase catalyzed reaction

The enzyme thymidylate synthase (TS) catalyzes a complex reaction that involves forming and breaking at least six covalent bonds. The physical nature of the hydride transfer step in this complex reaction cascade has been studied by means of isotope effects and their temperature dependence. Competitive kinetic isotope effects (KIEs) on the second-order rate constant (V/K) were measured over a temperature range of 5-45 degrees C. The observed H/T ((T)V/K(H)) and D/T ((T)V/K(D)) KIEs were used to calculate the intrinsic KIEs throughout the temperature range. The Swain-Schaad relationships between the H/T and D/T V/K KIEs revealed that the hydride transfer step is the rate-determining step at the physiological temperature of Escherichia coli (20-30 degrees C) but is only partly rate-determining at elevated and reduced temperatures. H/D KIE on the first-order rate constant k(cat) ((D)k = 3.72) has been previously reported [Spencer et al. (1997) Biochemistry 36, 4212-4222]. Additionally, the Swain-Schaad relationships between that (D)k and the V/K KIEs reported here suggested that at 20 degrees C the hydride transfer step is the rate-determining step for both rate constants. Intrinsic KIEs were calculated here and were found to be virtually temperature independent (DeltaE(a) = 0 within experimental error). The isotope effects on the preexponential Arrhenius factors for the intrinsic KIEs were A(H)/A(T) = 6.8 +/- 2.8 and A(D)/A(T) = 1.9 +/- 0.25. Both effects are significantly above the semiclassical (no-tunneling) predicted values and indicate a contribution of quantum mechanical tunneling to this hydride transfer reaction. Tunneling correction to transition state theory would predict that these isotope effects on activation parameters result from no energy of activation for all isotopes. Yet, initial velocity measurements over the same temperature range indicate cofactor inhibition and result in significant activation energy on k(cat) (4.0 +/- 0.1 kcal/mol). Taken together, the temperature-independent KIEs, the large isotope effects on the preexponential Arrhenius factors, and a significant energy of activation all suggest vibrationally enhanced hydride tunneling in the TS-catalyzed reaction.     

Role of the flexible loop of hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus in enzyme catalysis

The hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase), a type I PRTase, from Tritrichomonas foetus, is a potential target for antitritrichomonal chemotherapy. Structural data on all the type I PRTases reveal a highly flexible, 11-14-amino acid loop, presumably covering the active site. With the exception of a highly conserved Ser-Tyr dipeptide, the other amino acids constituting the loop vary widely among different PRTases. The roles of the conserved Ser73 and Tyr74 residues in the loop and the dynamics of the loop in T. foetus HGXPRTase were investigated using site-directed mutants, stop-flow kinetics, chemical modification, and two-dimensional (1)H-(15)N heteronuclear NMR relaxation experiments. S73A, Y74F, and Y74E mutants of HGXPRTase exhibited a 5-7-fold increase in K(m) for guanine and a 3-5-fold increase in K(m) for PRPP compared to that of the wild type, reflecting the decreased affinity of binding for the two substrates. The k(cat)'s for these mutant-catalyzed reactions, however, do not change appreciably from that of the wild-type enzyme. Stopped-flow fluorescence with a Y74W mutant showed no apparent quenching by adding either PRPP or GMP alone. When both PRPP and guanine were added together, however, the fluorescence was rapidly quenched, followed by a slow recovery as the enzyme-catalyzed reaction progressed, suggesting movement of the loop during catalysis. In the presence of 9-deazaguanine and PRPP, the rapidly quenched fluorescence was not recovered, suggesting a closed loop form. The accessibility of Trp74 in the flexible loop of the mutant enzyme was also analyzed using N-bromosuccinimide (NBS), which reacts specifically with the tryptophan residue. NBS reacted with the only tryptophan in the Y74W mutant enzyme and rendered the enzyme inactive. GMP or PRPP alone failed to protect the enzyme from NBS inactivation. However, the presence of both 9-deazaguanine and PPRP protected the enzyme, allowing it to retain up to 70% of its activity. An S75H mutant, labeled with [(15)N]histidine, was used in the (1)H-(15)N NMR study. Spectra obtained in the presence of enzyme substrates indicated an apparent stabilization of the loop only in the presence of 9-deazaguanine and PRPP. These experimental results thus clearly demonstrated stabilization of the flexible loop upon binding of both PRPP and guanine and suggested its involvement in enzyme catalysis.     

Multiple hydrogen kinetic isotope effects for enzymes catalyzing exchange with solvent: application to alanine racemase

Alanine racemase catalyzes the pyridoxal phosphate-dependent interconversion of the D- and L-isomers of alanine. Previous studies have shown that the enzyme employs a two-base mechanism in which Lys39 and Tyr265 are the acid/base catalysts. It is thus possible that stereoisomerization of the external aldimine intermediates occurs through a concerted double proton transfer without the existence of a distinct carbanionic intermediate. This possibility was tested by the application of multiple kinetic isotope effect (KIE) methodology to alanine racemase. The mutual dependence of primary substrate and solvent deuterium KIEs has been measured using equilibrium perturbation-type experiments. The conceptually straightforward measurement of the substrate KIE in H(2)O is complemented with a less intuitive protium washout perturbation-type measurement in D(2)O. The primary substrate KIE in the D --> L direction at 25 degrees C is reduced from 1.297 in H(2)O to 1.176 in D(2)O, while in the L --> D direction it is reduced from 1.877 in H(2)O to 1.824 in D(2)O. Similar reductions are also observed at 65 degrees C, the temperature to which the Bacillus stearothermophilus enzyme is adapted. These data strongly support a stepwise racemization of stereoisomeric aldimine intermediates in which a substrate-based carbanion is an obligatory intermediate. The ionizations observed in k(cat)/K(M) pH profiles have been definitively assigned based on the DeltaH(ion) values of the observed pK(a)'s with alanine and on the pH dependence of k(cat)/K(M) for the alternative substrate serine. The acidic pK(a) in the bell-shaped curve is due to the phenolic hydroxyl of Tyr265, which must be unprotonated for reaction with either isomer of alanine. The basic pK(a) is due to the substrate amino group, which must be protonated to react with Tyr265-unprotonated enzyme. A detailed reaction mechanism incorporating these results is proposed.     

Functional studies of active-site mutants from Drosophila melanogaster deoxyribonucleoside kinase. Investigations of the putative catalytic glutamate-arginine pair and of residues responsible for substrate specificity

The catalytic reaction mechanism and binding of substrates was investigated for the multisubstrate Drosophila melanogaster deoxyribonucleoside kinase. Mutation of E52 to D, Q and H plus mutations of R105 to K and H were performed to investigate the proposed catalytic reaction mechanism, in which E52 acts as an initiating base and R105 is thought to stabilize the transition state of the reaction. Mutant enzymes (E52D, E52H and R105H) showed a markedly decreased k(cat), while the catalytic activity of E52Q and R105K was abolished. The E52D mutant was crystallized with its feedback inhibitor dTTP. The backbone conformation remained unchanged, and coordination between D52 and the dTTP-Mg complex was observed. The observed decrease in k(cat) for E52D was most likely due to an increased distance between the catalytic carboxyl group and 5'-OH of deoxythymidine (dThd) or deoxycytidine (dCyd). Mutation of Q81 to N and Y70 to W was carried out to investigate substrate binding. The mutations primarily affected the K(m) values, whereas the k(cat) values were of the same magnitude as for the wild-type. The Y70W mutation made the enzyme lose activity towards purines and negative cooperativity towards dThd and dCyd was observed. The Q81N mutation showed a 200- and 100-fold increase in K(m), whereas k(cat) was decreased five- and twofold for dThd and dCyd, respectively, supporting a role in substrate binding. These observations give insight into the mechanisms of substrate binding and catalysis, which is important for developing novel suicide genes and drugs for use in gene therapy.     

Comparison of rates and kinetic isotope effects using PEG-modified variants and glycoforms of glucose oxidase: the relationship of modification of the protein envelope to C-H activation and tunneling

An earlier investigation of the temperature dependencies of rates and kinetic isotope effects (KIEs) in glucose oxidase (GO) used variants that differed in the extent of glycosylation at the surface of the protein. Kohen et al. [Kohen, A., Jonsson, T., and Klinman, J. P. (1997) Biochemistry 36, 2603-2611] presented evidence that the KIE on the Arrhenius prefactor varied as a function of protein modification, concluding that the degree of hydrogen tunneling at the active site was dependent on changes in mass at the surface. We now examine GO proteins containing polyethylene glycol (PEG) at their surface and a more extensively glycosylated form of GO, to distinguish simple mass effects from other sources of altered catalytic behavior. One PEG variant was created by modifying deglycosylated GO with short PEG chains (average of 350 Da each), while another contained a smaller number of long PEG chains (average of 5000 Da each). The light (146 kDa) and heavy (211 kDa) PEG variants and the hyperglycosylated variant display isotope effects on the Arrhenius prefactor that are similar (A(D)/A(T) = 0.55-0.62), while the unperturbed wild-type GO (WT-GO) is found to have an A(D)/A(T) that is reassessed as being close to unity. It appears that any modification of the protein surface away from that of the wild type gives rise to altered behavior for hydrogen transfer in the active site. We have also compared the effect of enthalpies of activation on both k(cat)/K(M) and k(cat) for the variants, introducing a new method to extract the k(cat)/K(M) rate constant and enthalpy of activation for the tritiated substrate from competitive KIE experiments. We find similar trends in Delta H(++) for both competitive and noncompetitive parameters and a smaller trend in k(cat) than reported earlier. Correlations are observed between A(D)/A(T) and both the enthalpies of activation and the thermal melt temperatures (T(M)) of the GO isoforms. In addition to the present study, there are now a number of examples where a perturbation of enzyme structure away from that of the wild type causes the observed KIE to become more temperature-dependent. The implications of these findings are discussed in the context of hydrogen tunneling and the relationship of protein structure and dynamics to this process.     

Half-of-sites binding of orotidine 5'-phosphate and alpha-D-5-phosphorylribose 1-diphosphate to orotate phosphoribosyltransferase from Saccharomyces cerevisiae supports a novel variant of the Theorell-Chance mechanism with alternating site catalysis

A ping-pong bi-bi kinetic mechanism ascribed to yeast orotate phosphoribosyltransferase (OPRTase) [Victor, J., Greenberg, L. B., and Sloan, D. L. (1979) J. Biol. Chem. 254, 2647-2655] has been shown to be inoperative [Witte, J. F., Tsou, R., and McClard, R. W. (1999) Arch. Biochem. Biophys. 361, 106-112]. Radiolabeled orotidine 5'-phosphate (OMP), generated in situ from [7-(14)C]-orotate and alpha-d-5-phoshorylribose 1-diphosphate (PRPP), binds tightly enough to OPRTase (a dimer composed of identical subunits) that the complex survives gel-filtration chromatography. When a sample of OMP.OPRTase is extensively dialyzed, a 1:1 (per OPRTase dimer) complex is detected by (31)P NMR. Titration of the apoenzyme with OMP yields a (31)P NMR spectrum with peaks for both free and enzyme-bound OMP when OMP is in excess; the complex maintains an OMP/enzyme ratio of 1:1 even when OMP is in substantial excess. A red shift in the UV spectrum of the OMP.OPRTase complex was exploited to measure K(d(OMP)) = 0.84 muM and to verify the 1:1 binding stoichiometry. PRPP forms a Mg(2+)-dependent 1:1 complex with the enzyme as observed by (31)P NMR. Isothermal titration calorimetry (ITC) experiments revealed 1:1 stoichiometries for both OMP and Mg(2+)-PRPP with OPRTase yielding K(d) values of 0.68 and 10 microM, respectively. The binding of either 1 equiv of OMP or PRPP is mutually exclusive. ITC experiments demonstrate that the binding of OMP is largely driven by increased entropy, suggesting substantial distal disordering of the protein. Analytical gel-filtration chromatography confirms that the OMP.OPRTase complex involves the dimeric form of enzyme. The off rate for release of OMP, determined by magnetization inversion transfer, was determined to be 27 s(-)(1). This off rate is somewhat less than the k(cat) in the biosynthetic direction (about 39 s(-)(1)); thus, the release of OMP from OMP.OPRTase may not be kinetically relevant to the steady-state reaction cycle. The body of available data can be explained in terms of alternating site catalysis with either a classical Theorell-Chance mechanism or, far more likely, a novel "double Theorell-Chance" mechanism unique to alternating site catalysis, leading us to propose co-temporal binding of orotate and the release of diphosphate as well as the binding of PRPP and the release of OMP that occur via ternary complexes in alternating site fashion across the two highly cooperative subunits of the enzyme. This novel "double Theorell-Chance" mechanism yields a steady-state rate equation indistinguishable in form from the observed classical ping-pong bi-bi kinetics.     

Mutational, structural, and kinetic evidence for a dissociative mechanism in the GDP-mannose mannosyl hydrolase reaction

GDP-mannose hydrolase (GDPMH) catalyzes the hydrolysis of GDP-alpha-d-sugars by nucleophilic substitution with inversion at the anomeric C1 atom of the sugar, with general base catalysis by H124. Three lines of evidence indicate a mechanism with dissociative character. First, in the 1.3 A X-ray structure of the GDPMH-Mg(2+)-GDP.Tris(+) complex [Gabelli, S. B., et al. (2004) Structure 12, 927-935], the GDP leaving group interacts with five catalytic components: R37, Y103, R52, R65, and the essential Mg(2+). As determined by the effects of site-specific mutants on k(cat), these components contribute factors of 24-, 100-, 309-, 24-, and >/=10(5)-fold, respectively, to catalysis. Both R37 and Y103 bind the beta-phosphate of GDP and are only 5.0 A apart. Accordingly, the R37Q/Y103F double mutant exhibits partially additive effects of the two single mutants on k(cat), indicating cooperativity of R37 and Y103 in promoting catalysis, and antagonistic effects on K(m). Second, the conserved residue, D22, is positioned to accept a hydrogen bond from the C2-OH group of the sugar undergoing substitution at C1, as was shown by modeling an alpha-d-mannosyl group into the sugar binding site. The D22A and D22N mutations decreased k(cat) by factors of 10(2.1) and 10(2.6), respectively, for the hydrolysis of GDP-alpha-d-mannose, and showed smaller effects on K(m), suggesting that the D22 anion stabilizes a cationic oxocarbenium transition state. Third, the fluorinated substrate, GDP-2F-alpha-d-mannose, for which a cationic oxocarbenium transition state would be destabilized by electron withdrawal, exhibited a 16-fold decrease in k(cat) and a smaller, 2.5-fold increase in K(m). The D22A and D22N mutations further decreased the k(cat) with GDP-2F-alpha-d-mannose to values similar to those found with GDP-alpha-d-mannose, and decreased the K(m) of the fluorinated substrate. The choice of histidine as the general base over glutamate, the preferred base in other Nudix enzymes, is not due to the greater basicity of histidine, since the pK(a) of E124 in the active complex (7.7) exceeded that of H124 (6.7), and the H124E mutation showed a 10(2.2)-fold decrease in k(cat) and a 4.0-fold increase in K(m) at pH 9.3. Similarly, the catalytic triad detected in the X-ray structure (H124- - -Y127- - -P120) is unnecessary for orienting H124, since the Y127F mutation had only 2-fold effects on k(cat) and K(m) with either H124 or E124 as the general base. Hence, a neutral histidine rather than an anionic glutamate may be necessary to preserve electroneutrality in the active complex.     

A novel engineered subtilisin BPN' lacking a low-barrier hydrogen bond in the catalytic triad

The low-barrier hydrogen bond (LBHB) between the Asp and His residues of the catalytic triad in a serine protease was perturbed via the D32C mutation in subtilisin BPN' (Bacillus protease N'). This mutant enzyme catalyzes the hydrolysis of N-Suc-Ala-Ala-Pro-Phe-SBzl with a k(cat)/K(m) value that is only 8-fold reduced from that of the wild-type (WT) enzyme. The value of k(cat)/K(m) for the corresponding p-nitroanilide (pNA) substrate is only 50-fold lower than that of the WT enzyme (DeltaDeltaG++ = 2.2 kcal/mol). The pK(a) controlling the ascending limb of the pH versus k(cat)/K(m) profile is lowered from 7.01 (WT) to 6.53 (D32C), implying that any hydrogen bond replacing that between Asp32 and His64 of the WT enzyme most likely involves the neutral thiol rather than the thiolate form of Cys32. It is shown by viscosity variation that the reaction of WT subtilisin with N-Suc-Ala-Ala-Pro-Phe-SBzl is 50% (sucrose) to 100% (glycerol) diffusion-controlled, while that of the D32C construct is 29% (sucrose) to 76% (glycerol) diffusion-controlled. The low-field NMR resonance of 18 ppm that has been assigned to a proton shared by Asp32 and His64, and is considered diagnostic of a LBHB in the WT enzyme, is not present in D32C subtilisin. Thus, the LBHB is not an inherent requirement for substantial rate enhancement for subtilisin.     

Rapid kinetic and isotopic studies on dialkylglycine decarboxylase

The two half-reactions of the pyridoxal 5'-phosphate (PLP)-dependent enzyme dialkylglycine decarboxylase (DGD) were studied individually by multiwavelength stopped-flow spectroscopy. Biphasic behavior was found for the reactions of DGD-PLP, consistent with two coexisting conformations observed in steady-state kinetics [Zhou, X., and Toney, M. D. (1998) Biochemistry 37, 5761--5769]. The half-reaction kinetic parameters depend on alkali metal ion size in a manner similar to that observed for steady-state kinetic parameters. The fast phase maximal rate constant for the 2-aminoisobutyrate (AIB) decarboxylation half-reaction with the potassium form of DGD-PLP is 25 s(-1), while that for the transamination half-reaction between DGD-PMP and pyruvate is 75 s(-1). The maximal rate constant for the transamination half-reaction of the potassium form of DGD-PLP with L-alanine is 24 s(-1). The spectral data indicate that external aldimine formation with either AIB or L-alanine and DGD-PLP is a rapid equilibrium process, as is ketimine formation from DGD-PMP and pyruvate. Absorption ascribable to the quinonoid intermediate is not observed in the AIB decarboxylation half-reaction, but is observed in the dead-time of the stopped-flow in the L-alanine transamination half-reaction. The [1-(13)C]AIB kinetic isotope effect (KIE) on k(cat) for the steady-state reaction is 1.043 +/- 0.003, while a value of 1.042 +/- 0.009 was measured for the AIB half-reaction. The secondary KIE measured for the AIB decarboxylation half-reaction with [C4'-(2)H]PLP is 0.92 +/- 0.02. The primary [2-(2)H]-L-alanine KIE on the transamination half-reaction is unity. Small but significant solvent KIEs are observed on k(cat) and k(cat)/K(M) for both substrates, and the proton inventories are linear in each case. NMR measurements of C2--H washout vs product formation give ratios of 105 and 14 with L-alanine and isopropylamine as substrates, respectively. These results support a rate-limiting, concerted C alpha-decarboxylation/C4'-protonation mechanism for the AIB decarboxylation reaction, and rapid equilibrium quinonoid formation followed by rate-limiting protonation to the ketimine intermediate for the L-alanine transamination half-reaction. Energy profiles for the two half-reactions are constructed.     

Chemical and mutagenic investigations of fatty acid amide hydrolase: evidence for a family of serine hydrolases with distinct catalytic properties.

Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme responsible for the catabolism of neuromodulatory fatty acid amides, including anandamide and oleamide. FAAH's primary structure identifies this enzyme as a member of a diverse group of alkyl amidases, known collectively as the "amidase signature family". At present, this enzyme family's catalytic mechanism remains poorly understood. In this study, we investigated the catalytic features of FAAH through mutagenesis, affinity labeling, and steady-state kinetic methods. In particular, we focused on the respective roles of three serine residues that are conserved in all amidase signature enzymes (S217, S218, and S241 in FAAH). Mutation of each of these serines to alanine resulted in a FAAH enzyme bearing significant catalytic defects, with the S217A and S218A mutants showing 2300- and 95-fold reductions in k(cat), respectively, and the S241A mutant exhibiting no detectable catalytic activity. The double S217A:S218A FAAH mutant displayed a 230 000-fold decrease in k(cat), supporting independent catalytic functions for these serine residues. Affinity labeling of FAAH with a specific nucleophile reactive inhibitor, ethoxy oleoyl fluorophosphonate, identified S241 as the enzyme's catalytic nucleophile. The pH dependence of FAAH's k(cat) and k(cat)/K(m) implicated a base involved in catalysis with a pK(a) of 7.9. Interestingly, mutation of each of FAAH's conserved histidines (H184, H358, and H449) generated active enzymes, indicating that FAAH does not contain a Ser-His-Asp catalytic triad commonly found in other mammalian serine hydrolytic enzymes. The unusual properties of FAAH identified here suggest that this enzyme, and possibly the amidase signature family as a whole, may hydrolyze amides by a novel catalytic mechanism.     

Mechanism of product chain length determination and the role of a flexible loop in Escherichia coli undecaprenyl-pyrophosphate synthase catalysis

The Escherichia coli undecaprayl-pyrophosphate synthase (UPPs) structure has been solved using the single wavelength anomalous diffraction method. The putative substrate-binding site is located near the end of the betaA-strand with Asp-26 playing a critical catalytic role. In both subunits, an elongated hydrophobic tunnel is found, surrounded by four beta-strands (betaA-betaB-betaD-betaC) and two helices (alpha2 and alpha3) and lined at the bottom with large residues Ile-62, Leu-137, Val-105, and His-103. The product distributions formed by the use of the I62A, V105A, and H103A mutants are similar to those observed for wild-type UPPs. Catalysis by the L137A UPPs, on the other hand, results in predominantly the formation of the C(70) polymer rather than the C(55) polymer. Ala-69 and Ala-143 are located near the top of the tunnel. In contrast to the A143V reaction, the C(30) intermediate is formed to a greater extent and is longer lived in the process catalyzed by the A69L mutant. These findings suggest that the small side chain of Ala-69 is required for rapid elongation to the C(55) product, whereas the large hydrophobic side chain of Leu-137 is required to limit the elongation to the C(55) product. The roles of residues located on a flexible loop were investigated. The S71A, N74A, or R77A mutants displayed 25-200-fold decrease in k(cat) values. W75A showed an 8-fold increase of the FPP K(m) value, and 22-33-fold increases in the IPP K(m) values were observed for E81A and S71A. The loop may function to bridge the interaction of IPP with FPP, needed to initiate the condensation reaction and serve as a hinge to control the substrate binding and product release.     

Enhancement, relaxation, and reversal of the stereoselectivity for phosphotriesterase by rational evolution of active site residues

The factors that govern the substrate reactivity and stereoselectivity of phosphotriesterase (PTE) toward organophosphotriesters containing various combinations of methyl, ethyl, isopropyl, and phenyl substituents at the phosphorus center were determined by systematic alterations in the dimensions of the active site. The wild type PTE prefers the S(P)-enantiomers over the corresponding R(P)-enantiomers by factors ranging from 10 to 90. Enlargement of the small subsite of PTE with the substitution of glycine and alanine residues for Ile-106, Phe-132, and/or Ser-308 resulted in significant improvements in k(cat)/K(a) for the R(P)-enantiomers of up to 2700-fold but had little effect on k(cat)/K(a) for the corresponding S(P)-enantiomers. The kinetic preferences for the S(P)-enantiomers were thus relaxed without sacrificing the inherent catalytic activity of the wild type enzyme. A reduction in the size of the large subsite with the mutant H257Y resulted in a reduction in k(cat)/K(a) for the S(P)-enantiomers, while the values of k(cat)/K(a) for the R(P)-enantiomers were essentially unchanged. The initial stereoselectivity observed with the wild type enzyme toward the chiral substrate library was significantly reduced with the H257Y mutant. Simultaneous alternations in the sizes of the large and small subsites resulted in the complete reversal of the chiral specificity. With this series of mutants, the R(P)-enantiomers were preferred as substrates over the corresponding S(P)-enantiomers by up to 500-fold. These results have demonstrated that the stereochemical determinants for substrate hydrolysis by PTE can be systematically altered through a rational reconstruction of the dimensions of the active site.     

Active site hydrophobic residues impact hydrogen tunneling differently in a thermophilic alcohol dehydrogenase at optimal versus nonoptimal temperatures

A growing body of data suggests that protein motion plays an important role in enzyme catalysis. Two highly conserved hydrophobic active site residues in the cofactor-binding pocket of ht-ADH (Leu176 and V260) have been mutated to a series of hydrophobic side chains of smaller size, as well as one deletion mutant, L176Δ. Mutations decrease k(cat) and increase K(M)(NAD(+)). Most of the observed decreases in effects on k(cat) at pH 7.0 are due to an upward shift in the optimal pH for catalysis; a simple electrostatic model is invoked that relates the change in pK(a) to the distance between the positively charged nicotinamide ring and bound substrate. Structural modeling of the L176Δ and V260A variants indicates the development of a cavity behind the nicotinamide ring without any significant perturbation of the secondary structure of the enzyme relative to that of the wild type. Primary kinetic isotope effects (KIEs) are modestly increased for all mutants. Above the dynamical transition at 30 °C for ht-ADH [Kohen, A., et al. (1999) Nature 399, 496], the temperature dependence of the KIE is seen to increase with a decrease in side chain volume at positions 176 and 260. Additionally, the relative trends in the temperature dependence of the KIE above and below 30 °C appear to be reversed for the cofactor-binding pocket mutants in relation to wild-type protein. The aggregate results are interpreted in the context of a full tunneling model of enzymatic hydride transfer that incorporates both protein conformational sampling (preorganization) and active site optimization of tunneling (reorganization). The reduced temperature dependence of the KIE in the mutants below 30 °C indicates that at low temperatures, the enzyme adopts conformations refractory to donor-acceptor distance sampling.     

Immobilized formaldehyde-metabolizing enzymes from Hansenula polymorpha for removal and control of airborne formaldehyde

Formaldehyde (FA)-containing indoor air has a negative effect on human health and should be removed by intensive ventilation or by catalytic conversion to non-toxic products. FA can be oxidized by alcohol oxidase (AOX) taking part in methanol metabolism of methylotrophic yeasts. In the present work, AOX isolated from a Hansenula polymorpha C-105 mutant (gcr1 catX) overproducing this enzyme in glucose medium, was tested for its ability to oxidize airborne FA. A continuous fluidized bed bioreactor (FBBR) was designed to enable an effective bioconversion of airborne FA by AOX or by permeabilized mutant H. polymorpha C-105 cells immobilized in calcium alginate beads. The immobilized AOX having a specific activity of 6-8 U mg?1 protein was shown to preserve 85-90% of the initial activity. The catalytic parameters of the immobilized enzyme were practically the same as for the free enzyme (k(cat)/K(m) was 2.35×103 M?1 s?1 vs 2.89×103 M?1 s?1, respectively). The results showed that upon bubbling of air containing from 0.3 up to 18.5 ppm FA through immobilized AOX in the range of 1.3-26.6 U g?1 of the gel resulted in essential decrease of FA concentration in the outlet gas phase (less than 0.02-0.03 ppm, i.e. 10-fold less than the threshold limit value). It was also demonstrated that a FBBR with immobilized permeabilized C-105 cells provided more than 90% elimination of airborne FA. The process was monitored by a specially constructed enzymatic amperometric biosensor based on FA oxidation by NAD+ and glutathione-dependent formaldehyde dehydrogenase from the recombinant H. polymorpha Tf 11-6 strain.