Structure and mechanism of action of an inverting mutant sialidase

Mutagenesis of the conserved tyrosine (Y370) of the Micromonospora viridifaciens sialidase to small amino acids changes the mechanism of catalysis from retention of anomeric configuration to inversion [Watson, J. N., et al. (2003) Biochemistry 42, 12682-12690]. For the Y370G mutant enzyme-catalyzed hydrolysis of a series of aryl sialosides and 3'-sialyllactose, the derived Br?nsted parameters (beta(lg)) on k(cat) and k(cat)/K(m) are -0.63 +/- 0.05 and -0.80 +/- 0.08, respectively. Thus, for the Y370G enzyme, glycosidic C-O bond cleavage is rate-determining. Analysis of the activity of the Y370G mutant and wild-type enzymes against a substrate [3,4-dihydro-2H-pyrano[3,2-c]pyridinium alpha-d-N-acetylneuraminide (DHP-alphaNeu5Ac)] whose hydrolysis cannot be accelerated by acid catalysis is consistent with these reactions proceeding via S(N)1 and S(N)2 mechanisms, respectively. The overall structure of the Y370G mutant sialidase active site is very similar to the previously reported wild-type structure [Gaskell, A., et al. (1995) Structure 3, 1197-1205], although removal of the tyrosine residue creates two significant changes to the active site. First, the anomeric oxygen atom of the hydrolysis product (beta-N-acetylneuraminic acid) and four water molecules bind in the large cavity created by the Y370G mutation. Second, the side chain of Asn310 moves to make a strong hydrogen bond to one of the bound water molecules.     

Mutagenesis of the conserved active-site tyrosine changes a retaining sialidase into an inverting sialidase

Mutagenesis of the conserved tyrosine (Y370) of the Micromonospora viridifaciens sialidase changes the mechanism of catalysis from retention of anomeric configuration to an unprecedented inverting mechanism in which water efficiently functions as the nucleophile. Three mutants, Y370A, Y370D, and Y370G, were produced recombinantly in Escherichia coli, and all are catalytically active against the activated substrate 4-methylumbelliferyl alpha-D-N-acetylneuraminide. The Y370D mutant was also shown to catalyze the hydrolysis of natural substrate analogues such as 3'-sialyllactose. A comparison of the pH-rate profiles for the wild-type and the Y370D mutant sialidase reveals no major differences, although with respect to the kinetic term k(cat)/K(m), an ionized form of the aspartate-370 enzyme is catalytically compromised. For the wild-type enzyme, the value of the Br?nsted parameter beta(lg) on k(cat) is 0.02 +/- 0.03, while for the Y370D mutant sialidase beta(lg) = -0.55 +/- 0.03 for the substrates with bad leaving groups. Thus, for the wild-type enzyme, a nonchemical step(s) is rate-limiting, but for the tyrosine mutant cleavage of the glycosidic C-O bond is rate-determining. The Br?nsted slopes derived for the kinetic parameter k(cat)/K(m) display a similar trend (beta(lg) -0.30 +/- 0.04 and -0.74 +/- 0.04 for the wild-type and Y370D, respectively). These results reveal that the tyrosine residue lowers the activation free energy for cleavage of 6'-sialyllactose, a natural substrate analogue, by more than 24.9 kJ mol(-1). Evidence is presented that the mutant sialidases operate by a dissociative mechanism, and the wild-type enzyme operates by a concerted mechanism.     

Leaving group dependence in the phosphorylation of Escherichia coli alkaline phosphatase by monophosphate esters

Values of kcat and Km have been measured for the Escherichia coli alkaline phosphatase catalyzed hydrolysis of 18 aryl and 12 alkyl monophosphate esters at pH 8.00 and 25 degrees C. A Br?nsted plot of log (kcat/Km) (M-1 s-1) vs. the pK of the leaving hydroxyl group exhibits two regression lines: log (kcat/Km) = -0.19 (+/- 0.02) pKArOH + 8.14 (+/- 0.15) log (kcat/Km) = -0.19 (+/- 0.01) pKROH + 5.89 (+/- 0.17) Alkyl phosphates with aryl or large lipophilic side chains are not correlated by the above equations and occupy positions intermediate between the two lines. The observed change in effective charge on the leaving oxygen of the ester (-0.2) is very small, consistent with substantial electrophilic participation of the enzyme with this atom. Cyclohexylammonium ion is a noncompetitive inhibitor against 4-nitrophenyl phosphate substrate at pH 8.00, and neutral phenol is a competitive inhibitor (Ki = 82.6 mM); these data and the 100-fold larger reactivity of aryl over alkyl esters are consistent with the existence of a lipophilic binding site for the leaving group of the substrate. The absence of a major steric effect in kcat/Km for substituted aryl esters confirms that the leaving group in the enzyme--substrate complex points away from the surface of the enzyme. Arguments are advanced to exclude a dissociative mechanism (involving a metaphosphate ion) for the enzyme-catalyzed substitution at phosphorus.     

Beta-glucosidase: substrate, solvent, and viscosity variation as probes of the rate-limiting steps

The second-order rate constants (kcat/Km) for the beta-glucosidase-catalyzed hydrolysis of aryl beta-D-glucopyranosides show a bell-shaped dependence of pH. The pKas that characterize this dependence are 4.4 (delta Hion approximately equal to 0) and 6.7 (delta Hion approximately equal to 0). In D2O these pKas are increased by 0.5 (+/- 0.1) unit, but there is no solvent isotope effect on the pH-independent second-order rate constant. Nath and Rydon [Nath, R. L., & Rydon, H. N. (1954) Biochem. J. 57, 1-10] examined the kinetics of the beta-glucosidase-catalyzed hydrolysis of a series of substituted phenyl glucosides. We have extended this study to include glucosides with phenol leaving groups of pKa less than 7. Br?nsted plots for this extended series were nonlinear for both kcat/Km and kcat. Br?nsted coefficients for those compounds with leaving groups of pKa greater than 7 (for kcat/Km) or pKa greater than 8.5 (for kcat) were nearly equal to -1.0, indicating substantial negative charge buildup on the leaving group in the transition state. The nonlinearity indicates an intermediate in the reaction. This was confirmed by partitioning experiments in the presence of methanol as a competing glucose acceptor. A constant product ratio, [methyl glucoside]/[glucose], was found with aryl glucoside substrates varying over 16,000-fold in reactivity (V/K), indicative of a common intermediate. Viscosity variation (in sucrose-containing buffers) was used to probe the extent to which the beta-glucosidase reactions are diffusion-controlled.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental charge measurement at leaving oxygen in the bovine ribonuclease A catalyzed cyclization of uridine 3'-phosphate aryl esters

The title esters are demonstrated to be specific substrates of bovine pancreatic ribonuclease A (EC 3.1.27.5). The Br?nsted dependence of kcat/Km at pH 7.50 for the enzyme-catalyzed cyclization versus the pKa of the leaving phenol exhibits two regression lines of almost identical slope for respectively 2-chlorophenols and 2,6-unsubstituted phenols: log kcat/Km = -0.20 pKa ArOH + 5.47 (n = 5, r = 0.957); log kcat/Km = -0.17 pKa ArOH + 5.79 (n = 4, r = 0.965). Comparison of the Br?nsted beta 1g's with that for the standard reaction where imidazole catalyzes the cyclization (beta 1g = -0.59) indicates considerably less development of negative charge on the leaving oxygen in the enzyme case, providing experimental evidence for the hypothesis that electrophilic assistance is involved in catalysis. The existence of two essentially parallel Br?nsted correlations is not reflected in the standard reaction of substrate with imidazole. Modeling studies indicate that the phenyl ring of the substrate can take up a range of positions away from the active site; the presence of ortho chloro substituents considerably restricts the motion of the phenyl leaving group.     

Contribution of the active site aspartic acid to catalysis in the bacterial neuraminidase from Micromonospora viridifaciens

A recombinant D92G mutant sialidase from Micromonospora viridifaciens has been cloned, expressed and purified. Kinetic studies reveal that the replacement of the conserved aspartic acid with glycine results in a catalytically competent retaining sialidase that possesses significant activity against activated substrates. The contribution of this aspartate residue to the free energy of hydrolysis for natural substrates is greater than 19 kJ/mol. The three dimensional structure of the D92G mutant shows that the removal of aspartic acid 92 causes no significant re-arrangement of the active site, and that an ordered water molecule substitutes for the carboxylate group of D92.     

Identification of the catalytic residues in family 52 glycoside hydrolase,a beta-xylosidase from Geobacillus stearothermophilus T-6

beta-d-Xylosidases (EC 3.2.1.37) are exo-type glycoside hydrolases that hydrolyze short xylooligosaccharides to xylose units. The enzymatic hydrolysis of the glycosidic bond involves two carboxylic acid residues, and their identification, together with the stereochemistry of the reaction, provides crucial information on the catalytic mechanism. Two catalytic mutants of a beta-xylosidase from Geobacillus stearothermophilus T-6 were subjected to detailed kinetic analysis to verify their role in catalysis. The activity of the E335G mutant decreased approximately 106-fold, and this activity was enhanced 103-fold in the presence of external nucleophiles such as formate and azide, resulting in a xylosyl-azide product with an opposite anomeric configuration. These results are consistent with Glu335 as the nucleophile in this retaining enzyme. The D495G mutant was subjected to detailed kinetic analysis using substrates bearing different leaving groups (pKa). The mutant exhibited 103-fold reduction in activity, and the Br?nsted plot of log(kcat) versus pKa revealed that deglycosylation is the rate-limiting step, indicating that this step was reduced by 103-fold. The rates of the glycosylation step, as reflected by the specificity constant (kcat/Km), were similar to those of the wild type enzyme for hydrolysis of substrates requiring little protonic assistance (low pKa) but decreased 102-fold for those that require strong acid catalysis (high pKa). Furthermore, the pH dependence profile of the mutant enzyme revealed that acid catalysis is absent. Finally, the presence of azide significantly enhanced the mutant activity accompanied with the generation of a xylosyl-azide product with retained anomeric configuration. These results are consistent with Asp495 acting as the acid-base in XynB2.     

Detailed comparative analysis of the catalytic mechanisms of beta-N-acetylglucosaminidases from families 3 and 20 of glycoside hydrolases

Beta-N-acetylglucosaminidases are commonly occurring enzymes involved in the degradation of polysaccharides and glycoconjugates containing N-acetylglucosamine residues. Such enzymes have been classified into glycoside hydrolase families 3 and 20 and are believed to follow distinct chemical mechanisms. Family 3 enzymes are thought to follow a standard retaining mechanism involving a covalent glycosyl enzyme intermediate while family 20 enzymes carry out a substrate-assisted mechanism involving the transient formation of an enzyme-sequestered oxazoline or oxazolinium ion intermediate. Detailed mechanistic analysis of representatives of these two families provides support for these mechanisms as well as detailed insights into transition state structure. Alpha-secondary deuterium kinetic isotope effects of kH/kD = 1.07 and 1.10 for Streptomyces plicatus beta-hexosaminidase (SpHex) and Vibrio furnisii beta-N-acetylglucosaminidase (ExoII) respectively indicate transition states with oxocarbenium ion character in each case. Br?nsted plots for hydrolysis of a series of aryl hexosaminides are quite different in the two cases. For SpHex a large degree of proton donation is suggested by the relatively low value of beta(lg) (-0.29) on kcat/Km, compared with a beta(lg) of -0.79 for ExoII. Most significantly the Taft plots derived from kinetic parameters for a series of p-nitrophenyl N-acyl glucosaminides bearing differing levels of fluorine substitution in the N-acyl group are completely different. A very strong dependence (slope = -1.29) is seen for SpHex, indicating direct nucleophilic participation by the acetamide, while essentially no dependence (0.07) is seen for ExoII, suggesting that the acetamide plays purely a binding role. Taken together these data provide unprecedented insight into enzymatic glycosyl transfer mechanisms wherein the structures of both the nucleophile and the leaving group are systematically varied.     

Mechanism of GlvA from Bacillus subtilis: a detailed kinetic analysis of a 6-phospho-alpha-glucosidase from glycoside hydrolase family 4

GlvA, a 6-phospho-alpha-glucosidase from Bacillus subtilis assigned to glycoside hydrolase family 4, catalyzes the hydrolysis of maltose 6'-phosphate via a redox-elimination-addition mechanism requiring NAD+ as cofactor. In contrast to previous reports and consistent with the proposed mechanism, GlvA is only activated in the presence of the nicotinamide cofactor in its oxidized, and not the reduced NADH, form. Significantly, GlvA catalyzes the hydrolysis of both 6-phospho-alpha- and 6-phospho-beta-glucosides containing activated leaving groups such as p-nitrophenol and does so with retention and inversion, respectively, of anomeric configuration. Mechanistic details of the individual bond cleaving and forming steps were probed using a series of 6-phospho-alpha- and 6-phospho-beta-glucosides. Primary deuterium kinetic isotope effects (KIEs) were measured for both classes of substrates in which either the C2 or the C3 protons have been substituted with a deuterium, consistent with C-H bond cleavage at each center being partially rate-limiting. Kinetic parameters were also determined for 1-[2H]-substituted substrates, and depending on the substrates and the reaction conditions, the measurements of kcat and kcat/KM produced either no KIEs or inverse KIEs. In conjunction with results of Br?nsted analyses with both aryl 6-phospho-alpha- and beta-glucosides, the kinetic data suggest that GlvA utilizes an E1cb mechanism analogous to that proposed for the Thermotoga maritima BglT, a 6-phospho-beta-glucosidase in glycoside hydrolase family 4 (Yip, V.L.Y et al. (2006) Biochemistry 45, 571-580). The pattern of isotope effects measured and the observation of very similar kcat values for all substrates, including unactivated and natural substrates, indicate that the oxidation and deprotonation steps are rate-limiting steps in essentially all cases. This mechanism permits the cleavage of both alpha- and beta-glycosides within the same active site motif and, for activated substrates that do not require acid catalysis for cleavage, within the same active site, yielding the product sugar-6-phosphate in the same anomeric form in the two cases.     

Human cathepsin H: deletion of the mini-chain switches substrate specificity from aminopeptidase to endopeptidase

The mini-chain of human cathepsin H has been identified as the major structural element determining the protease's substrate specificity. A genetically engineered mutant of human cathepsin H lacking the mini-chain, des[Glu(-18)-Thr(-11)]-cathepsin H, exhibits endopeptidase activity towards the synthetic substrate Z-Phe-Arg-NH-Mec (kcat = 0.4 s(-1), Km = 92 microM, kcat/Km = 4348 M(-1) s(-1)) which is not cleaved by r-wt cathepsin H. However, the mutant enzyme shows only minimal aminopeptidase activity for H-Arg-NH-Mec (kcat = 0.8 s(-1), Km = 3.6 mM, kcat/Km = 222 M(-1) s(-1)) which is one of the best known substrates for native human cathepsin H (kcat = 2.5 s(-1), Km = 150 microM, kcat/Km = 16666 M(-1) s(-1)). Inhibition studies with chicken egg white cystatin and E-64 suggest that the mini-chain normally restricts access of inhibitors to the active site. The kinetic data on substrates hydrolysis and enzyme inhibition point out the role of the mini-chain as a structural framework for transition state stabilization of free alpha-amino groups of substrates and as a structural barrier for endopeptidase-like substrate cleavage.     

Probing the transition-state structure of dual-specificity protein phosphatases using a physiological substrate mimic

Dual-specificity phosphatases (DSPs) belong to the large family of protein tyrosine phosphatases that contain the active-site motif (H/V)CxxGxxR(S/T), but unlike the tyrosine-specific enzymes, DSPs are able to catalyze the efficient hydrolysis of both phosphotyrosine and phosphoserine/threonine found on signaling proteins, as well as a variety of small-molecule aryl and alkyl phosphates. It is unclear how DSPs accomplish similar reaction rates for phosphoesters, whose reactivity (i.e., pK(a) of the leaving group) can vary by more than 10(8). Here, we utilize the alkyl phosphate m-nitrobenzyl phosphate (mNBP), leaving-group pK(a) = 14.9, as a physiological substrate mimic to probe the mechanism and transition state of the DSP, Vaccinia H1-related (VHR). Detailed pH and kinetic isotope effects of the V/K value for mNBP indicates that VHR reacts with the phosphate dianion of mNBP and that the nonbridge phosphate oxygen atoms are unprotonated in the transition state. (18)O and solvent isotope effects indicate differences in the respective timing of the proton transfer to the leaving group and P-O fission; with the alkyl ester substrate, protonation is ahead of P-O fission, while with the aryl substrate, the two processes are more synchronous. Kinetic analysis of the general-acid mutant D92N with mNBP was consistent with the requirement of Asp-92 in protonating the ester oxygen, either in a step prior to significant P-O bond cleavage or in a concerted but asynchronous mechanism in which protonation is ahead of P-O bond fission. Collectively, the data indicate that VHR and likely all DSPs can match leaving-group potential with the timing of the proton transfer to the ester oxygen, such that diverse aryl and alkyl phosphoesters are turned over with similar catalytic efficiency.     

Bacterial and viral sialidases: contribution of the conserved active site glutamate to catalysis

Mutagenesis of the conserved glutamic acid of influenza type A (E277) and Micromonospora viridifaciens (E260) sialidases was performed to probe the contribution of this strictly conserved residue to catalysis. Kinetic studies of the E260D and E260C M. viridifaciens mutant enzymes reveal that the overall mechanism of action has not changed. That is, the mutants are retaining sialidases in which glycosylation and deglycosylation are rate-limiting for k(cat)/K(m) and k(cat), respectively. The solvent kinetic isotope effect and proton inventory on k(cat) for the E260C mutant sialidase provide strong evidence that the newly installed cysteine residue provides little catalytic acceleration. The results are consistent with the conserved aspartic acid residue (D92) becoming the key general acid/base residue in the catalytic cycle. In addition, the E277D mutant influenza type A sialidase is catalytically active toward 4-nitrophenyl α-D-sialoside, although no measurable hydrolysis of natural substrates was observed. Thus, mutating the glutamate residue (E277) to an aspartate increases the activation free energy of hydrolysis for natural substrates by >22 kJ/mol.     

Mutagenesis and mechanistic study of a glycoside hydrolase family 54 alpha-L-arabinofuranosidase from Trichoderma koningii

A GH (glycoside hydrolase) family 54 alpha-L-arabinofuranosidase from Trichoderma koningii G-39 (termed Abf) was successfully expressed in Pichia pastoris and purified to near homogeneity by cation-exchange chromatography. To determine the amino acid residues essential for the catalytic activity of Abf, extensive mutagenesis of 24 conserved glutamate and aspartate residues was performed. Among the mutants, D221N, E223Q and D299N were found to decrease catalytic activity significantly. The kcat values of the D221N and D299N mutants were 7000- and 1300-fold lower respectively, than that of the wild-type Abf. E223Q was nearly inactive. These results are consistent with observations obtained from the Aspergillus kawachii alpha-L-arabinofuranosidase three-dimensional structure. This structure indicates that Asp221 of T. koningii Abf is significant for substrate binding and that Glu223 as well as Asp299 function as a nucleophile and a general acid/base catalyst for the enzymatic reaction respectively. The catalytic mechanism of wild-type Abf was further investigated by NMR spectroscopy and kinetic analysis. The results showed that Abf is a retaining enzyme. It catalyses the hydrolysis of various substrates via the formation of a common intermediate that is probably an arabinosyl-enzyme intermediate. A two-step, double-displacement mechanism involving first the formation, and then the breakdown, of an arabinosyl-enzyme intermediate was proposed. Based on the kcat values of a series of aryl-alpha-L-arabinofuranosides catalytically hydrolysed by wild-type Abf, a relatively small Br?nsted constant, beta(lg)=-0.18, was obtained, suggesting that the rate-limiting step of the enzymatic reaction is the dearabinosylation step. Further kinetic studies with the D299G mutant revealed that the catalytic activity of this mutant depended largely on the pK(a) values (>6) of leaving phenols, with beta(lg)=-1.3, indicating that the rate-limiting step of the reaction becomes the arabinosylation step. This kinetic outcome supports the idea that Asp299 is the general acid/base residue. The pH activity profile of D299N provided further evidence strengthening this suggestion.     

Detailed dissection of a new mechanism for glycoside cleavage: alpha-1,4-glucan lyase

The unusual enzyme, Gracilariopsis alpha-1,4-glucan lyase of the sequence-related glycoside hydrolase family 31, cleaves the glycosidic bond of alpha-1,4-glucans via a beta-elimination reaction involving a covalent glycosyl-enzyme intermediate (Lee, S. S., Yu, S., and Withers, S. G. (2002) J. Am. Chem. Soc. 124, 4948-4949). The classical bell-shaped pH dependence of k(cat)/K(m) indicates two ionizable groups in the active site with apparent pK(a) values of 3.05 and 6.66. Br?nsted relationships of log k(cat) versus pK(a) and log(k(cat)/K(m)) versus pK(a) for a series of aryl glucosides both show a linear monotonic dependence on leaving group pK(a) with low beta(lg) values of 0.32 and 0.33, respectively. The combination of these low beta(lg) values with large secondary deuterium kinetic isotope effects (k(H)/k(D) = 1.16 - 1.19) on the first step indicate a glycosylation step with substantial glycosidic bond cleavage and proton donation to the leaving group oxygen at the transition state. Developed oxocarbenium ion character of the transition state is also suggested by the potent inhibition afforded by acarbose and 1-deoxynojirimycin (K(i) = 20 and 130 nM, respectively) and by the substantial rate reduction afforded by adjacent fluorine substitution. For only one substrate, 5-fluoro-alpha-D-glucopyranosyl fluoride, was the second elimination step shown to be rate-limiting. The large alpha-secondary deuterium kinetic isotope effect (k(H)/k(D) = 1.23) at C-1 and the small primary deuterium kinetic isotope effect (k(H)/k(D) = 1.92) at C-2 confirm an E2 mechanism with strong E1 character for this second step. This considerable structural and mechanistic similarity with retaining alpha-glucosidases is clear evidence for the evolution of an enzyme mechanism within the family.     

Leaving group dependence and proton inventory studies of the phosphorylation of a cytoplasmic phosphotyrosyl protein phosphatase from bovine heart.

The kcat and Km values for the bovine heart low molecular weight phosphotyrosyl protein phosphatase catalyzed hydrolysis of 16 aryl phosphate monoesters and of five alkyl phosphate monoesters having the structure Ar(CH2)nOPO3H2 (n = 1-5) were measured at pH 5.0 and 37 degrees C. With the exception of alpha-naphthyl phosphate and 2-chlorophenyl phosphate, which are subject to steric effects, the values of kcat are effectively constant for the aryl phosphate monoesters. This is consistent with the catalysis being nucleophilic in nature, with the existence of a common covalent phosphoenzyme intermediate, and with the breakdown of this intermediate being rate-limiting. In contrast, kcat for the alkyl phosphate monoesters is much smaller and the rate-limiting step for these substrates is interpreted to be the phosphorylation of the enzyme. A single linear correlation is observed for a plot of log (kcat/Km) vs leaving group pKa for both classes of substrates at pH 5.0: log (kcat/Km) = -0.28pKa + 6.88 (n = 19, r = 0.89), indicating a uniform catalytic mechanism for the phosphorylation event. The small change in effective charge (-0.28) on the departing oxygen of the substrate is similar to that observed in the specific acid catalyzed hydrolysis of monophosphate monoanions (-0.27) and is consistent with a strong electrophilic interaction of the enzyme with this oxygen atom in the transition state. The D2O solvent isotope effect and proton inventory experiments indicate that only one proton is "in flight" in the transition state of the phosphorylation process and that this proton transfer is responsible for the reduction of effective charge on the leaving oxygen.     

Do enzymes change the nature of transition states? Mapping the transition state for general acid-base catalysis of a serine protease

The properties of the transition state for serine protease-catalyzed hydrolysis of an amide bond were determined for a series of subtilisin variants from Bacillus lentus. There is no significant change in the structure of the enzyme upon introduction of charged mutations S156E/S166D, suggesting that changes in catalytic activity reflect global properties of the enzyme. The effect of charged mutations on the pK(a) of the active site histidine-64 N(epsilon)(2)-H was correlated with changes in the second-order rate constant k(cat)/K(m) for hydrolysis of tetrapeptide anilides at low ionic strength with a Br?nsted slope alpha = 1.1. The solvent isotope effect (D)2(O)(k(cat)/K(m))(1) = 1.4 +/- 0.2. These results are consistent with a rate-limiting breakdown of the tetrahedral intermediate in the acylation step with hydrogen bond stabilization of the departing amine leaving group. There is an increase in the ratio of hydrolysis of succinyl-Ala-Ala-Pro-Phe-anilides for p-nitroaniline versus aniline leaving groups with variants with more basic active site histidines that can be described by the interaction coefficient p(xy) = delta beta(lg)/delta pK(a) (H64) = 0.15. This is attributed to increased hydrogen bonding of the active site imidazolium N-H to the more basic amine leaving group as well as electrostatic destabilization of the transition state. A qualitative characterization of the transition state is presented in terms of a reaction coordinate diagram that is defined by the structure-reactivity parameters.     

Structure-activity relationships in the hydrolysis of substrates by the phosphotriesterase from Pseudomonas diminuta

The mechanism and substrate specificity of the phosphotriesterase from Pseudomonas diminuta have been examined. The enzyme hydrolyzes a large number of phosphotriester substrates in addition to paraoxon (diethyl p-nitrophenyl phosphate) and its thiophosphate analogue, parathion. The two ethyl groups in paraoxon can be changed to propyl and butyl groups, but the maximal velocity and Km values decrease substantially. The enzyme will not hydrolyze phosphomonoesters or -diesters. There is a linear correlation between enzymatic activity and the pKa of the phenolic leaving group for 16 paraoxon analogues. The beta value in the corresponding Br?nsted plot is -0.8. No effect on either Vmax or Vmax/Km is observed when sucrose is used to increase the relative solvent viscosity by 3-fold. These results are consistent with rate-limiting phosphorus-oxygen bond cleavage. A plot of log V versus pH for the hydrolysis of paraoxon shows one enzymatic group that must be unprotonated for activity with a pKa of 6.1. The deuterium isotope effect by D2O on Vmax and Vmax/Km is 2.4 and 1.2, respectively, and the proton inventory is linear, which indicates that only one proton is "in flight" during the transition state. The inhibition patterns by the products are consistent with a random kinetic mechanism.     

Mutations at the guanosine-binding site of the Tetrahymena ribozyme also affect site-specific hydrolysis.

Self-splicing group I introns use guanosine as a nucleophile to cleave the 5' splice site. The guanosine-binding site has been localized to the G264-C311 base pair of the Tetrahymena intron on the basis of analysis of mutations that change the specificity of the nucleophile from G (guanosine) to 2AP (2-aminopurine ribonucleoside) (F. Michel et al. (1989) Nature 342, 391-395). We studied the effect of these mutations (G-U, A-C and A-U replacing G264-C311) in the L-21 ScaI version of the Tetrahymena ribozyme. In this enzymatic system (kcat/Km)G monitors the cleavage step. This kinetic parameter decreased by at least 5 x 10(3) when the G264-C311 base pair was mutated to an A-U pair, while (kcat/Km)2AP increased at least 40-fold. This amounted to an overall switch in specificity of at least 2 x 10(5). The nucleophile specificity (G > 2AP for the G-C and G-U pairs, 2AP > G for the A-U and A-C pairs) was consistent with the proposed hydrogen bond between the nucleotide at position 264 and N1 of the nucleophile. Unexpectedly, the A-U and A-C mutants showed a decrease of an order of magnitude in the rate of ribozyme-catalyzed hydrolysis of RNA, in which H2O or OH- replaces G as the nucleophile, whereas the G-U mutant showed a decrease of only 2-fold. The low hydrolysis rates were not restored by raising the Mg2+ concentration or lowering the temperature. In addition, the mutant ribozymes exhibited a pattern of cleavage by Fe(II)-EDTA indistinguishable from that of the wild type, and the [Mg2+]1/2 for folding of the A-U mutant ribozyme was the same as that of the wild type. Therefore the guanosine-binding site mutations do not appear to have a major effect on RNA folding or stability. Because changing G264 affects the hydrolysis reaction without perturbing the global folding of the RNA, we conclude that the catalytic role of this conserved nucleotide is not limited to guanosine binding.     

The hydrolase and transferase activity of an inverting mutant sialidase using non-natural beta-sialoside substrates

The Y370G inverting mutant sialidase from Micromonospora viridifaciens possesses beta-sialidase activity with phenyl beta-sialoside (Ph-betaNeuAc) to give alpha-sialic acid as the first formed product. The derived catalytic rate constants for k(cat) and k(cat)/K(m) are 13.3 +/- 0.3 and (2.9 +/- 0.3) x 10(5) M(-)(1) s(-)(1), respectively. This enzyme is highly specific for the phenyl substrate, with substituted phenyl and thiophenyl leaving groups having k(cat) values that are at least 1000-fold lower. In addition, the Y370G mutant can transfer the sialic acid moiety from Ph-betaNeuAc to lactose in yields of up to 13%. Greater than 90% of the sialyl-lactose product formed in the coupling reactions is the alpha-2,6-isomer. A library encoding 6 x 10(5) different sialidases was constructed by mutating Y370, E260, T309, N310, and N311, residues that include and are proximal the catalytic tyrosine residue. A total of 2628 individuals were screened for hydrolytic activity against 4-nitrophenyl 2-thio-beta-sialoside and 4-methylumbelliferyl beta-sialoside. However, none of the mutants screened possessed a significant activity against either of the beta-sialosides.     

Effects of deuterium substitution alpha and beta to the reaction centre, 18O substitution in the leaving group, and aglycone acidity on hydrolyses of aryl glucosides and glucosyl pyridinium ions by yeast alpha-glucosidase. A probable failure of the antiperiplanar-lone-pair hypothesis in glycosidase catalysis.

Neither kcat. nor kcat./Km for five aryl alpha-D-glucopyranosides correlates with aglycone pKa, and isotope effects, described according to the convention used by Cleland [(1982) CRC Crit. Rev. Biochem. 13, 385-428], of 18(V) = 1.002 +/- 0.008, alpha D(V) = 1.01 +/- 0.04 and alpha D(V/K) = 0.969 +/- 0.035 are observed for p-nitrophenyl, and one of beta D(V) = 1.02 +/- 0.04 for phenyl alpha-D-glucopyranoside; kcat. but not kcat./Km, correlates with aglycone pKa for five alpha-D-glucopyranosyl pyridinium ions with a Brønsted coefficient of -0.61 +/- 0.06, and isotope effects of alpha D(V) = 1.22 +/- 0.02, beta D(V) = 1.13 +/- 0.01 and alpha D(V/K) = 1.018 +/- 0.046 for the 4-bromoisoquinolinium, and alpha D(V) = 1.15 +/- 0.02 and beta D(V) = 1.085 +/- 0.011 for the pyridinium salts are observed. These data require that a non-covalent event, fast in the case of the N-glycosides but slow in the case of the O-glycosides, precedes bond-breaking, and that bond-breaking involves substantial charge development on the glycone and near-perpendicularity of the C2-H bond to the planar oxocarbonium ion system. A model meeting these requirements is that the non-covalent event is a conjoint change of protein and substrate conformation which puts the pyranose ring in the 2,5B conformation of the bond-breaking transition state. This model also explains the contrast between the powerful inhibition of the enzyme by deoxynojirimycin (Ki = 23 +/- 3 microM) and feeble inhibition by castanospermine [Saul, Chambers, Molyneux & Elbein (1983) Arch. Biochem. Biophys. 221, 593-597], but is directly contrary to the predictions of Deslongchamps'' ''Theory of Stereoelectronic Control'' [Deslongchamps (1975) Tetrahedron 31, 2463-2490; (1983) Stereoelectronic Effects in Organic Chemistry, p. 39, Pergamon Press, Oxford].