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.     

Kinetic analyses of divalent cation-dependent EcoRV digestions on a DNA-immobilized quartz crystal microbalance

Enzymatic digestion with a type IIP restriction endonuclease EcoRV was investigated on a DNA-immobilized 27-MHz quartz crystal microbalance (QCM). Real-time observations of both the enzyme binding process and the DNA cleavage process of EcoRV were followed by frequency (mass) changes on the QCM, which were dependent on divalent cations such as Ca(2+) or Mg(2+). In the presence of Ca(2+), the site-specific binding of EcoRV to DNA could be observed, without the catalytic process. On the other hand, in the presence of Mg(2+), both the binding of the enzyme to the specific DNA (mass increase) and the site-specific cleavage reaction (mass decrease) could be observed continuously from QCM frequency changes. From time courses of frequency (mass) changes, each kinetic parameter, namely binding rate constants (k(on)), dissociation rate constants (k(off)), dissociation constants (K(d)) of EcoRV to DNA, and catalytic rate constant (k(cat)) of the cleavage reaction, could be determined. The binding kinetic parameters of EcoRV in the presence of Ca(2+) were consistent with those of the binding process followed by the cleavage process in the presence of Mg(2+). The k(cat) value obtained by the QCM method was also consistent with that obtained by other methods. This study is the first to simultaneously determine k(on), k(off), and k(cat) for a type IIP restriction endonuclease on one device.     

Engineered monomeric human histidine triad nucleotide-binding protein 1 hydrolyzes fluorogenic acyl-adenylate and lysyl-tRNA synthetase-generated lysyl-adenylate

Hint1 is a homodimeric protein and member of the ubiquitous HIT superfamily. Hint1 catalyzes the hydrolysis of purine phosphoramidates and lysyl-adenylate generated by lysyl-tRNA synthetase (LysRS). To determine the importance of homodimerization on the biological and catalytic activity of Hint1, the dimer interface of human Hint1 (hHint1) was destabilized by replacement of Val(97) of hHint1 with Asp, Glu, or Arg. The mutants were shown to exist as monomers in solution by a combination of size exclusion chromatograph, static light scattering, and chemically induced dimerization studies. Circular dichroism studies revealed little difference between the stability of the V97D, V97E, and wild-type hHint1. Relative to wild-type and the V97E mutant, however, significant perturbation of the V97D mutant structure was observed. hHint1 was shown to prefer 3-indolepropionic acyl-adenylate (AIPA) over tryptamine adenosine phosphoramidate monoester (TpAd). Wild-type hHint1 was found to be 277- and 1000-fold more efficient (k(cat)/K(m) values) than the V97E and V97D mutants, respectively. Adenylation of wild-type, V97D, and V97E hHint1 by human LysRS was shown to correlate with the mutant k(cat)/K(m) values using 3-indolepropionic acyl-adenylate as a substrate, but not tryptamine adenosine phosphoramidate monoester. Significant perturbations of the active site residues were not detected by molecular dynamics simulations of the hHint1s. Taken together, these results demonstrate that for hHint1; 1) the efficiency (k(cat)/K(m)) of acylated AMP hydrolysis, but not maximal catalytic turnover (k(cat)), is dependent on homodimerization and 2) the hydrolysis of lysyl-AMP generated by LysRS is not dependent on homodimerization if the monomer structure is similar to the wild-type structure.     

New insights from the structure-function analysis of the catalytic region of human platelet phosphodiesterase 3A: a role for the unique 44-amino acid insert

Human phosphodiesterase 3A (PDE3A) degrades cAMP, the major inhibitor of platelet function, thus potentiating platelet function. Of the 11 human PDEs, only PDE3A and 3B have 44-amino acid inserts in the catalytic domain. Their function is not clear. Incubating Sp-adenosine-3',5'-cyclic-S-(4-bromo-2,3-di-oxobutyl) monophosphorothioate (Sp-cAMPS-BDB) with PDE3A irreversibly inactivates the enzyme. High pressure liquid chromatography (HPLC) analysis of a tryptic digest yielded an octapeptide within the insert of PDE3A ((K)T(806)YNVTDDK(813)), suggesting that a substrate-binding site exists within the insert. Because Sp-cAMPS-BDB reacts with nucleophilic residues, mutants Y807A, D811A, and D812A were produced. Sp-cAMPS-BDB inactivates D811A and D812A but not Y807A. A docking model showed that Tyr(807) is 3.3 angstroms from the reactive carbon, whereas Asp(811) and Asp(812) are >15 angstroms away from Sp-cAMPS-BDB. Y807A has an altered K(m) but no change in k(cat). Activity of wild type but not Y807A is inhibited by an anti-insert antibody. These data suggest that Tyr(807) is modified by Sp-cAMPS-BDB and involved in substrate binding. Because the homologous amino acid in PDE3B is Cys(792), we prepared the mutant Y807C and found that its K(m) and k(cat) were similar to the wild type. Moreover, Sp-cAMPS-BDB irreversibly inactivates Y807C with similar kinetics to wild type, suggesting that the tyrosine may, like the cysteine, serve as a H donor. Kinetic analyses of nine additional insert mutants reveal that H782A, T810A, Y814A, and C816S exhibit an altered k(cat) but not K(m), indicating that catalysis is modulated. We document a new functional role for the insert in which substrate binding may produce a conformational change. This change would allow the substrate to bind to Tyr(807) and other amino acids in the insert to interact with residues important for catalysis in the active site cleft.     

The role of factor XIa (FXIa) catalytic domain exosite residues in substrate catalysis and inhibition by the Kunitz protease inhibitor domain of protease nexin 2

To select residues in coagulation factor XIa (FXIa) potentially important for substrate and inhibitor interactions, we examined the crystal structure of the complex between the catalytic domain of FXIa and the Kunitz protease inhibitor (KPI) domain of a physiologically relevant FXIa inhibitor, protease nexin 2 (PN2). Six FXIa catalytic domain residues (Glu(98), Tyr(143), Ile(151), Arg(3704), Lys(192), and Tyr(5901)) were subjected to mutational analysis to investigate the molecular interactions between FXIa and the small synthetic substrate (S-2366), the macromolecular substrate (factor IX (FIX)) and inhibitor PN2KPI. Analysis of all six Ala mutants demonstrated normal K(m) values for S-2366 hydrolysis, indicating normal substrate binding compared with plasma FXIa; however, all except E98A and K192A had impaired values of k(cat) for S-2366 hydrolysis. All six Ala mutants displayed deficient k(cat) values for FIX hydrolysis, and all were inhibited by PN2KPI with normal values of K(i) except for K192A, and Y5901A, which displayed increased values of K(i). The integrity of the S1 binding site residue, Asp(189), utilizing p-aminobenzamidine, was intact for all FXIa mutants. Thus, whereas all six residues are essential for catalysis of the macromolecular substrate (FIX), only four (Tyr(143), Ile(151), Arg(3704), and Tyr(5901)) are important for S-2366 hydrolysis; Glu(98) and Lys(192) are essential for FIX but not S-2366 hydrolysis; and Lys(192) and Tyr(5901) are required for both inhibitor and macromolecular substrate interactions.     

Direct monitoring of allosteric recognition of type IIE restriction endonuclease EcoRII

EcoRII is a homodimer with two domains consisting of a DNA-binding N terminus and a catalytic C terminus and recognizes two specific sequences on DNA. It shows a relatively complicated cleavage reaction in bulk solution. After binding to either recognition site, EcoRII cleaves the other recognition site of the same DNA (cis-binding) strand and/or the recognition site of the other DNA (trans-binding) strand. Although it is difficult to separate these two reactions in bulk solution, we could simply obtain the binding and cleavage kinetics of only the cis-binding by following the frequency (mass) changes of a DNA-immobilized quartz-crystal microbalance (QCM) responding to the addition of EcoRII in aqueous solution. We obtained the maximum binding amounts (Deltam(max)), the dissociation constants (K(d)), the binding and dissociation rate constants (k(on) and k(off)), and the catalytic cleavage reaction rate constants (k(cat)) for wild-type EcoRII, the N-terminal-truncated form (EcoRII N-domain), and the mutant derivatives in its C-terminal domain (K263A and R330A). It was determined from the kinetic analyses that the N-domain, which covers the catalytic C-domain in the absence of DNA, preferentially binds to the one DNA recognition site while transforming EcoRII into an active form allosterically, and then the secondary C-domain binds to and cleaves the other recognition site of the DNA strand.     

The mechanism of DNA cytosine-5 methylation. Kinetic and mutational dissection of Hhai methyltransferase

Kinetic and binding studies involving a model DNA cytosine-5-methyltransferase, M.HhaI, and a 37-mer DNA duplex containing a single hemimethylated target site were applied to characterize intermediates on the reaction pathway. Stopped-flow fluorescence studies reveal that cofactor S-adenosyl-l-methionine (AdoMet) and product S-adenosyl-l-homocysteine (AdoHcy) form similar rapidly reversible binary complexes with the enzyme in solution. The M.HhaI.AdoMet complex (k(off) = 22 s(-)1, K(D) = 6 microm) is partially converted into products during isotope-partitioning experiments, suggesting that it is catalytically competent. Chemical formation of the product M.HhaI.(Me)DNA.AdoHcy (k(chem) = 0.26 s(-)1) is followed by a slower decay step (k(off) = 0.045 s(-)1), which is the rate-limiting step in the catalytic cycle (k(cat) = 0.04 s(-)1). Analysis of reaction products shows that the hemimethylated substrate undergoes complete (>95%) conversion into fully methylated product during the initial burst phase, indicating that M.HhaI exerts high binding selectivity toward the target strand. The T250N, T250D, and T250H mutations, which introduce moderate perturbation in the catalytic site, lead to substantially increased K(D)(DNA(ternary)), k(off)(DNA(ternary)), K(M)(AdoMet(ternary)) values but small changes in K(D)(DNA(binary)), K(D)(AdoMet(binary)), k(chem), and k(cat). When the target cytosine is replaced with 5-fluorocytosine, the chemistry step leading to an irreversible covalent M.HhaI.DNA complex is inhibited 400-fold (k(chem)(5FC) = 0.7 x 10(-)3 s(-)1), and the Thr-250 mutations confer further dramatic decrease of the rate of the covalent methylation k(chem). We suggest that activation of the pyrimidine ring via covalent addition at C-6 is a major contributor to the rate of the chemistry step (k(chem)) in the case of cytosine but not 5-fluorocytosine. In contrast to previous reports, our results imply a random substrate binding order mechanism for M.HhaI.     

Substrate and inhibitor specificity of Mycobacterium avium dihydrofolate reductase

Dihydrofolate reductase (EC 1.5.1.3) is a key enzyme in the folate biosynthetic pathway. Information regarding key residues in the dihydrofolate-binding site of Mycobacterium avium dihydrofolate reductase is lacking. On the basis of previous information, Asp31 and Leu32 were selected as residues that are potentially important in interactions with dihydrofolate and antifolates (e.g. trimethoprim), respectively. Asp31 and Leu32 were modified by site-directed mutagenesis, giving the mutants D31A, D31E, D31Q, D31N and D31L, and L32A, L32F and L32D. Mutated proteins were expressed in Escherichia coli BL21(DE3)pLysS and purified using His-Bind resin; functionality was assessed in comparison with the recombinant wild type by a standard enzyme assay, and growth complementation and kinetic parameters were evaluated. All Asp31 substitutions affected enzyme function; D31E, D31Q and D31N reduced activity by 80-90%, and D31A and D31L by > 90%. All D31 mutants had modified kinetics, ranging from three-fold (D31N) to 283-fold (D31L) increases in K(m) for dihydrofolate, and 12-fold (D31N) to 223 077-fold (D31L) decreases in k(cat)/K(m). Of the Leu32 substitutions, only L32D caused reduced enzyme activity (67%) and kinetic differences from the wild type (seven-fold increase in K(m); 21-fold decrease in k(cat)/K(m)). Only minor variations in the K(m) for NADPH were observed for all substitutions. Whereas the L32F mutant retained similar trimethoprim affinity as the wild type, the L32A mutation resulted in a 12-fold decrease in affinity and the L32D mutation resulted in a seven-fold increase in affinity for trimethoprim. These findings support the hypotheses that Asp31 plays a functional role in binding of the substrate and Leu32 plays a functional role in binding of trimethoprim.     

Complete reversal of coenzyme specificity of xylitol dehydrogenase and increase of thermostability by the introduction of structural zinc

Pichia stipitis NAD(+)-dependent xylitol dehydrogenase (XDH), a medium-chain dehydrogenase/reductase, is one of the key enzymes in ethanol fermentation from xylose. For the construction of an efficient biomass-ethanol conversion system, we focused on the two areas of XDH, 1) change of coenzyme specificity from NAD(+) to NADP(+) and 2) thermostabilization by introducing an additional zinc atom. Site-directed mutagenesis was used to examine the roles of Asp(207), Ile(208), Phe(209), and Asn(211) in the discrimination between NAD(+) and NADP(+). Single mutants (D207A, I208R, F209S, and N211R) improved 5 approximately 48-fold in catalytic efficiency (k(cat)/K(m)) with NADP(+) compared with the wild type but retained substantial activity with NAD(+). The double mutants (D207A/I208R and D207A/F209S) improved by 3 orders of magnitude in k(cat)/K(m) with NADP(+), but they still preferred NAD(+) to NADP(+). The triple mutant (D207A/I208R/F209S) and quadruple mutant (D207A/I208R/F209S/N211R) showed more than 4500-fold higher values in k(cat)/K(m) with NADP(+) than the wild-type enzyme, reaching values comparable with k(cat)/K(m) with NAD(+) of the wild-type enzyme. Because most NADP(+)-dependent XDH mutants constructed in this study decreased the thermostability compared with the wild-type enzyme, we attempted to improve the thermostability of XDH mutants by the introduction of an additional zinc atom. The introduction of three cysteine residues in wild-type XDH gave an additional zinc-binding site and improved the thermostability. The introduction of this mutation in D207A/I208R/F209S and D207A/I208R/F209S/N211R mutants increased the thermostability and further increased the catalytic activity with NADP(+).     

Change in substrate preference of Streptomyces aminopeptidase through modification of the environment around the substrate binding site

We attempted to alter the substrate preference of aminopeptidase from Streptomyces septatus TH-2 (SSAP). Because Asp198 and Phe221 of SSAP are located in the substrate binding site, we screened 2,000 mutant enzymes with D198X/F221X mutations. By carrying out this examination, we obtained two enzymes; one specifically hydrolyzed an arginyl derivative, and the other specifically hydrolyzed a cystinyl derivative (65- and 12.5-fold higher k(cat) values for hydrolysis of p-nitroanilide derivatives than those of the wild type, respectively).     

Determination of steady-state kinetic parameters for a xylanase-catalyzed hydrolysis of neutral underivatized xylooligosaccharides by mass spectrometry

A direct mass spectrometric approach was used for the determination of steady-state kinetic parameters, the turnover number (k(cat)), the Michaelis constant (K(M)), and the specificity constant (k(cat)/K(M)) for an enzyme-catalyzed hydrolysis of xylooligosaccharides. Electrospray ionization mass spectrometry was performed to observe product distributions and to determine k(cat), K(M), and k(cat)/K(M) values for Trichoderma reesei endo-1,4-beta-xylanase II (TRX II) with xylohexaose (Xyl(6)), xylopentaose (Xyl(5)), xylotetraose (Xyl(4)), and xylotriose (Xyl(3)) as substrates. The determined k(cat)/K(M) values (0.93, 0.37, 0.027, and 0.00015 microM(-1) s(-1), respectively) indicated that Xyl(6) was the most preferred substrate of TRX II. In addition, the obtained K(M) value for Xyl(5) (136 microM) was roughly twice as high as that for Xyl(6) (73 microM), suggesting that at least six putative subsites contribute to the substrate binding in the active site of TRX II. Previous mass spectrometric assays for enzyme kinetics have been used mostly in the case of reactions that result in a transfer of acidic groups (e.g., phosphate) into neutral oligosaccharides giving rise to negatively charged products. Here we demonstrate that such analysis is also feasible in the case of neutral underivatized oligosaccharides. Implications of the results for the catalytic mechanism of TRX II in particular are discussed.     

Substrate-dependent competency of the catalytic triad of prolyl oligopeptidase

Prolyl oligopeptidase, a serine peptidase unrelated to trypsin and subtilisin, is implicated in memory disorders and is an important target of drug design. The catalytic competence of the Asp(641) residue of the catalytic triad (Ser(554), Asp(641), His(680)) was studied using the D641N and D641A variants of the enzyme. Both variants displayed 3 orders of magnitude reduction in k(cat)/K(m) for benzyloxycarbonyl-Gly-Pro-2-naphthylamide. Using an octapeptide substrate, the decrease was 6 orders of magnitude, whereas with Z-Gly-Pro-4-nitrophenyl ester there was virtually no change in k(cat)/K(m). This indicates that the contribution of Asp(641) is very much dependent on the substrate-leaving group, which was not the case for the classic serine peptidase, trypsin. The rate constant for benzyloxycarbonyl-Gly-Pro-thiobenzylester conformed to this series as demonstrated by a method designed for monitoring the hydrolysis of thiolesters in the presence of thiol groups. Alkylation of His(680) with Z-Gly-Pro-CH(2)Cl was concluded with similar rate constants for wild-type and D641A variant. However, kinetic measurements with Z-Gly-Pro-OH, a product-like inhibitor, indicated that the His(680) is not accessible in the enzyme variants. Crystal structure determination of these mutants revealed subtle perturbations related to the catalytic activity. Many of these observations show differences in the catalysis between trypsin and prolyl oligopeptidase.     

Mutational analysis of a feruloyl esterase from Aspergillus awamori involved in substrate discrimination and pH dependence

We cloned the feruloyl esterase A gene from Aspergillus awamori (AwfaeA) and engineered it to study substrate specificity and pH dependence of catalysis. Based on the crystal structures of two type-A feruloyl esterases (FAE-III and AnFAEA) from Aspergillus niger, residues located in the flap region of AwFAEA (Asp71, Thr72, Asp77, and Tyr80) were replaced with corresponding amino acid residues (Ile, Arg, Asn, and Phe), respectively, found in the lid of lipases from Rhizomucor miehei (RmLIP) and Humicola lanuginose (HlLIP). Furthermore, Asp77 of AwFAEA, which is conserved in Aspergillus FAEs and lipases, was replaced with a hydrophobic residue (Ile). Kinetic analysis of the mutant enzymes showed that the higher catalytic efficiency of the D77I and Y80F mutants toward alpha-naphthylbutyrate (C4) and alpha-naphthylcaprylate (C8), respectively, was due to a lower K(m) value. The higher catalytic efficiency of D77N toward C4 substrate was due to a combination of decreased K(m) and considerably increased k(cat). The D71I and Y80F mutants showed some activity toward long-acyl chain esters. On the other hand, the D77I mutant had no detectable activity toward phenolic acid methyl esters and feruloylated arabinoxylan. Moreover, the pH optima of the D77I, D77N, and Y80F mutants increased from 5.0 to 7.0-8.0, 7.0, and 6.0, respectively.     

Characterization of the functional role of Asp141, Asp194, and Asp464 residues in the Mn2+-L-malate binding of pigeon liver malic enzyme

Pigeon liver malic enzyme was inactivated and cleaved at Asp141, Asp194, and Asp464 by the Cu2+-ascorbate system in acidic environment. Site-specific mutagenesis was performed at these putative metal-binding sites. Three point mutants, D141N, D194N, and D464N; three double mutants, D(141,194)N, D(194,464)N, and D(141,464)N; and a triple mutant, D(141,194,464)N; as well as the wild-type malic enzyme (WT) were successfully cloned and expressed in Escherichia coli cells. All recombinant enzymes, except the triple mutant, were purified to apparent homogeneity by successive Q-Sepharose and adenosine-2',5'-bisphosphate-agarose columns. The mutants showed similar apparent Km,NADP values to that of the WT. The Km,Mal value was increased in the D141N and D194N mutants. The Km,Mn value, on the other hand, was increased only in the D141N mutant by 14-fold, corresponding to approximately 1.6 kcal/mol for the Asp141-Mn2+ binding energy. Substrate inhibition by L-malate was only observed in WT, D464N, and D(141,464)N. Initial velocity experiments were performed to derive the various kinetic parameters. The possible interactions between Asp141, Asp194, and Asp464 were analyzed by the double-mutation cycles and triple-mutation box. There are synergistic weakening interactions between Asp141 and Asp194 in the metal binding that impel the D(141,194)N double mutant to an overall specificity constant [k(cat)/(Kd,Mn Km,Mal Km,NADP)] at least four orders of magnitude smaller than the WT value. This difference corresponds to an increase of 6.38 kcal/mol energy barrier for the catalytic efficiency. Mutation at Asp464, on the other hand, has partial additivity on the mutations at Asp141 and Asp194. The overall specificity constants for the double mutants D(194,464)N and D(141,464)N or the triple mutant D(141,194,464)N were decreased by only 10- to 100-fold compared to the WT. These results strongly suggest the involvement of Asp141 in the Mn2+-L-malate binding for the pigeon liver malic enzyme. The Asp194 and Asp464, which may be oxidized by nonspecific binding of Cu2+, are involved in the Mn2+-L-malate binding or catalysis indirectly by modulating the binding affinity of Asp141 with the Mn2+.     

The bifunctional active site of s-adenosylmethionine synthetase. Roles of the active site aspartates.

S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPP(i)) from ATP, and subsequently PPP(i) is hydrolyzed to PP(i) and P(i) before product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues. Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg(2+) ions (* denotes a residue from a second subunit); aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers. However, all have k(cat) reductions of approximately 10(3)-fold in AdoMet synthesis, whereas the MgATP and methionine K(m) values change by less than 3- and 8-fold, respectively. In the partial reaction of PPP(i) hydrolysis, mutants of the Mg(2+) binding residues have >700-fold reduced catalytic efficiency (k(cat)/K(m)), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPP(i) hydrolysis by Mg(2+) site mutants is improved by AdoMet, like the wild type enzyme. In contrast AdoMet reduces the catalytic efficiency for PPP(i) hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca(2+) uniquely activates the D271A mutant enzyme to 15% of the level of Mg(2+), in contrast to the approximately 1% Ca(2+) activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca(2+) and the smaller Mg(2+).     

Butyrylcholinesterase-catalyzed hydrolysis of N-methylindoxyl acetate: analysis of volume changes upon reaction and hysteretic behavior

Hydrolysis of the neutral substrate N-methylindoxyl acetate (NMIA) by wild-type human butyrylcholinesterase (BuChE) and peripheral site mutants (D70G, Y332A, D70G/Y332A) was found to follow the Michaelis-Menten kinetics. K(m) was 0.14 mM for wild-type, and 0.07-0.16 mM for D70G, Y332A and D70G/Y332A, indicating that the peripheral site is not involved in NMIA binding. The values of k(cat) were of the same order for all enzymes: 12,000-18,000 min(-1). Volume changes upon substrate binding (-DeltaV(K(m))) and the activation volumes (DeltaV++(k(cat)) associated with hydrolysis of NMIA were calculated from the pressure dependence of the catalytic constants. Values of -DeltaV(K(m)) indicate that NMIA binds to an aromatic residue, presumed to be W82, the active site binding locus. Binding is accompanied by a release of water molecules from the gorge. Residue 70 controls the number of water molecules that are released upon substrate binding. The values of DeltaV++(k(cat)), which are positive for wild-type and faintly positive for D70G, clearly indicate that the catalytic steps are accompanied by re-entry of water into the gorge. Results support the premise that residue D70 is involved in the conformational stabilization of the active site gorge and in control of its hydration. A slow transient, preceding the steady state, was seen on a time scale of several minutes. The induction time rapidly increased with NMIA concentration to reach a limit at substrate saturation. Much shorter induction times (<1 min) were seen for hydrolysis of benzoylcholine (BzCh) by wild-type BuChE and for hydrolysis of butyrylthiocholine (BuSCh) by the active site mutants E197Q and E197Q/G117H. This slow transient was interpreted in terms of hysteresis without kinetic cooperativity. The hysteretic behavior of BuChE results from a slow conformational equilibrium between two enzyme states E and E'. NMIA binds only to the primed form E'. Kosmotropic salts and hydrostatic pressure were found to shift the equilibrium toward E'. The E-->E' transition is accompanied by a negative activation volume (DeltaV++(0)= -45+/-10 ml/mol), and the E' form is more compact than E. Hydration water in the gorge of E' appears to be more structured than in the unprimed form.     

Diversity in the oxidation of substrates by cytochrome P450 2D6: lack of an obligatory role of aspartate 301-substrate electrostatic bonding

Cytochrome P450 (P450) 2D6 was first identified as the polymorphic human debrisoquine hydroxylase and subsequently shown to catalyze the oxidation of a variety of drugs containing a basic nitrogen. Residue Asp301 has been characterized as being involved in electrostatic interactions with substrates on the basis of homology modeling and site-directed mutagenesis experiments [Ellis, S. W., Hayhurst, G. P., Smith, G., Lightfoot, T., Wong, M. M. S., Simula, A. P., Ackland, M. J., Sternberg, M. J. E., Lennard, M. S., Tucker, G. T., and Wolf, C. R. (1995) J. Biol. Chem. 270, 29055-29058]. However, pharmacophore models based on the role of Asp301 in substrate binding are compromised by reports of catalytic activity toward substrates devoid of a basic nitrogen, which have generally been ignored. We characterized a high-affinity ligand for P450 2D6, also devoid of a basic nitrogen atom, spirosulfonamide [4-[3-(4-fluorophenyl)-2-oxo-1-oxaspiro[4.4]non-3-en-4-yl]benzenesulfonamide], with K(s) 1.6 microM. Spirosulfonamide is a substrate for P450 2D6 (k(cat) 6.5 min(-)(1) for the formation of a syn spiromethylene carbinol, K(m) 7 microM). Mutation of Asp301 to neutral residues (Asn, Ser, Gly) did not substantially affect the binding of spirosulfonamide (K(s) 2.5-3.5 microM). However, the hydroxylation of spirosulfonamide was attenuated in these mutants to the same extent (90%) as for the classic nitrogenous substrate bufuralol, and the effect of the D301N substitution was manifested on k(cat) but not K(m). Analogues of spirosulfonamide were also evaluated as ligands and substrates. Analogues in which the sulfonamide moiety was modified to an amide, thioamide, methyl sulfone, or hydrogen were ligands with K(s) values of 1.7-32 microM. All were substrates, and the methyl sulfone analogue was oxidized to the syn spiromethylene carbinol analogue of the major spirosulfonamide product. The D301N mutation produced varying changes in the oxidation patterns of the spirosulfonamide analogues. The peptidometic ritonavir and the steroids progesterone and testosterone had been reported to be substrates for P450 2D6, but the affinities (K(s)) were unknown; these were estimated to be 1.2, 1.5, and 15 microM, respectively (cf. 6 microM for the classic substrate bufuralol). The results are consistent with a role of Asp301 other than electrostatic interaction with a positively charged ligand. H-Bonding or electrostatic interactions probably enhance binding of some substrates, but our results show that it is not required for all substrates and explain why predictive models fail to recognize the proclivity for many substrates, especially those containing no basic nitrogen.     

Ancient adaptation of the active site of tryptophanyl-tRNA synthetase for tryptophan binding

The amino acid binding domains of the tryptophanyl (TrpRS)- and tyrosyl-tRNA synthetases (TyrRS) of Bacillus stearothermophilus are highly homologous. These similarities suggest that conserved residues in TrpRS may be responsible for both determining tryptophan recognition and discrimination against tyrosine. This was investigated by the systematic mutation of TrpRS residues based upon the identity of homologous positions in TyrRS. Of the four residues which interact directly with the aromatic side chain of tryptophan (Phe5, Met129, Asp132, and Val141) replacements of Asp132 led to significant changes in the catalytic efficiency of Trp aminoacylation (200-1250-fold reduction in k(cat)/K(M)) and substitution of Val141 by the larger Glu side chain reduced k(cat)/K(M) by 300-fold. Mutation of Pro127, which determines the position of active-site residues, did not significantly effect Trp binding. Of the mutants tested, D132N TrpRS also showed a significant reduction in discrimination against Tyr, with Tyr acting as a competitive inhibitor but not a substrate. The analogous residue in B. stearothermophilusTyrRS (Asp176) has also been implicated as a determinant of amino acid specificity in earlier studies [de Prat Gay, G., Duckworth, H. W., and Fersht, A. R. (1993) FEBS Lett. 318, 167-171]. This striking similarity in the function of a highly conserved residue found in both TrpRS and TyrRS provides mechanistic support for a common origin of the two enzymes.     

Engineering E. coli Alkaline Phosphatase Yields Changes of Catalytic Activity,Thermal Stability and Phosphate Inhibition

To investigate the function of aspartic acid residue 101 and arginine residue 166 in the active site of Escherichia coli alkaline phosphatase (EAP), two single mutants D101S (Asp 101 &#77 Ser) and R166K (Arg 166 &#77 Lys) and a double mutant D101S/R166K of EAP were generated through site-directed mutagenesis based on over-lap PCR method. Their enzymatic kinetic properties, thermal stabilities and possible reaction mechanism were explored. In the presence of inorganic phosphate acceptor, 1 M diethanolamine buffer, the k cat for D101S mutant enzyme increased 10-fold compared to that of wild-type EAP. The mutant R166K has a 2-fold decrease of k cat relative to the wild-type EAP, but the double mutant D101S/R166K was in the middle of them, indicative of an additive effect of these two mutations. On the other hand, the catalytic efficiencies of mutant enzymes are all reduced because of a substantial increase of K m values. All three mutants were more resistant to phosphate inhibitor than the wild-type enzyme. The analysis of the kinetic data suggests that (1) the D101S mutant enzyme obtains a higher catalytic activity by allowing a faster release of the product; (2) the R166K mutant enzyme can reduce the binding of the substrate and phosphate competitive inhibitor; (3) the double mutant enzyme has characteristics of both quicker catalytic turnover number and decreased affinity for competitive inhibitor. Additionally, pre-steady-state kinetics of D101S and D101S/R166K mutants revealed a transient burst followed by a linear steady state phase, obviously different from that of wild-type EAP, suggesting that the rate-limiting step has partially change from the release of phosphate from non-covalent E-Pi complex to the hydrolysis of covalent E-Pi complex for these two mutants.     

Michaelis constants of mushroom tyrosinase with respect to oxygen in the presence of monophenols and diphenols

The complex reaction mechanism of tyrosinase involves three enzymatic forms, two overlapping catalytic cycles and a dead-end complex. Analytical expressions for the catalytic and Michaelis constants of tyrosinase towards phenols and oxygen were derived for both, monophenolase and diphenolase activities of the enzyme. Thus, the Michaelis constants of tyrosinase towards the oxygen (K(mO(2))) are related with the respective catalytic constants for monphenols (k(M)(cat)) and o-diphenols (k(D)(cat)), as well as with the rate constant, k(+8). We recently determined the experimental value of the rate constant for the binding of oxygen to deoxytyrosinase (k(+8)) by stopped-flow assays. In this paper, we calculate theoretical values of K(mO(2)) from the experimental values of catalytic constants and k(+8) towards several monophenols and o-diphenols. The reliability and the significance of the values of K(mO(2)) are discussed.