Site-directed mutagenesis of active site residues of phosphite dehydrogenase

Phosphite dehydrogenase (PTDH) catalyzes the unusual oxidation of phosphite to phosphate with the concomitant reduction of NAD(+) to NADH. PTDH shares significant amino acid sequence similarity with D-hydroxy acid dehydrogenases (DHs), including strongly conserved catalytic residues His292, Glu266, and Arg237. Site-directed mutagenesis studies corroborate the essential role of His292 as all mutants of this residue were completely inactive. Histidine-selective inactivation studies with diethyl pyrocarbonate provide further evidence regarding the importance of His292. This residue is most likely the active site base that deprotonates the water nucleophile. Kinetic analysis of mutants in which Arg237 was changed to Leu, Lys, His, and Gln revealed that Arg237 is involved in substrate binding. These results agree with the typical role of this residue in D-hydroxy acid DHs. However, Glu266 does not play the typical role of increasing the pK(a) of His292 to enhance substrate binding and catalysis as the Glu266Gln mutant displayed an increased k(cat) and unchanged pH-rate profile compared to those of wild-type PTDH. The role of Glu266 is likely the positioning of His292 and Arg237 with which it forms hydrogen bonds in a homology model. Homology modeling suggests that Lys76 may also be involved in substrate binding, and this postulate is supported by mutagenesis studies. All mutants of Lys76 display reduced activity with large effects on the K(m) for phosphite, and Lys76Cys could be chemically rescued by alkylation with 2-bromoethylamine. Whereas a positively charged residue is absolutely essential for activity at the position of Arg237, Lys76 mutants that lacked a positively charged side chain still had activity, indicating that it is less important for binding and catalysis. These results highlight the versatility of nature's catalytic scaffolds, as a common framework with modest changes allows PTDH to catalyze its unusual nucleophilic displacement reaction and d-hydroxy acid DHs to oxidize alcohols to ketones.     

Pre-steady-state studies of phosphite dehydrogenase demonstrate that hydride transfer is fully rate limiting

Phosphite dehydrogenase (PTDH) is a unique NAD-dependent enzyme that catalyzes the oxidation of inorganic phosphite to phosphate. The enzyme has great potential for cofactor regeneration, and mechanistic studies have provided some insight into the residues that are important for catalysis. In this investigation, pre-steady-state studies were performed on the His6-tagged wild-type (WT) enzyme, several active site mutants, a thermostable mutant (12X-PTDH), and a thermostable mutant with dual cofactor specificity (NADP-12X-PTDH). Stopped-flow kinetic experiments indicate that slow steps after hydride transfer do not significantly limit the rate of reaction for the WT enzyme, the active site mutants, or the thermostable mutant. Pre-steady-state kinetic isotope effects (KIEs) and single-turnover experiments further confirm that slow steps after the chemical step do not significantly limit the rate of reaction for any of these proteins. Collectively, these results suggest that the hydride transfer step is fully rate determining in PTDH and that the observed KIE on kcat is the intrinsic effect in WT PTDH and the mutants examined. In contrast, a slow step after catalysis may partially limit the rate of phosphite oxidation by NADP-12X-PTDH with NADP as the cofactor. Finally, site-directed mutagenesis of Asp79 indicates that this residue is important in orienting Arg237 for proper interaction with phosphite.     

Inhibition and pH dependence of phosphite dehydrogenase

Phosphite dehydrogenase (PTDH) catalyzes the NAD-dependent oxidation of phosphite to phosphate, a reaction that is 15 kcal/mol exergonic. The enzyme belongs to the family of D-hydroxy acid dehydrogenases. Five other family members that were analyzed do not catalyze the oxidation of phosphite, ruling out the possibility that this is a ubiquitous activity of these proteins. PTDH does not accept any alternative substrates such as thiophosphite, hydrated aldehydes, and methylphosphinate, and potential small nucleophiles such as hydroxylamine, fluoride, methanol, and trifluoromethanol do not compete with water in the displacement of the hydride from phosphite. The pH dependence of k(cat)/K(m,phosphite) is bell-shaped with a pK(a) of 6.8 for the acidic limb and a pK(a) of 7.8 for the basic limb. The pK(a) of 6.8 is assigned to the second deprotonation of phosphite. However, whether the dianionic form of phosphite is the true substrate is not clear since a reverse protonation mechanism is also consistent with the available data. Unlike k(cat)/K(m,phosphite), k(cat) and k(cat)/K(m,NAD) are pH-independent. Sulfite is a strong inhibitor of PTDH that is competitive with respect to phosphite and uncompetitive with respect to NAD(+). Incubation of the enzyme with NAD(+) and low concentrations of sulfite results in a covalent adduct between NAD(+) and sulfite in the active site of the enzyme that binds very tightly. Fluorescent titration studies provided the apparent dissociation constants for NAD(+), NADH, sulfite, and the sulfite-NAD(+) adduct. Substrate isotope effect studies with deuterium-labeled phosphite resulted in small normal isotope effects (1.4-2.1) on both k(cat) and k(cat)/K(m,phosphite) at pH 7.25 and 8.0. Solvent isotope effects (SIEs) on k(cat) are similar in size; however, the SIE of k(cat)/K(m,phosphite) at pH 7.25 is significantly larger (4.4), whereas at pH 8.0, it is the inverse (0.6). The pH-rate profile of k(cat)/K(m,phosphite), which predicts that the observed SIEs will have a significant thermodynamic origin, can account for these effects.     

Coenzyme specificity of human monomeric carbonyl reductase: contribution of Lys-15, Ala-37 and Arg-38

Short-chain dehydrogenases/reductases catalyze the oxidoreduction of alcohol and carbonyl compounds using either NAD or NADPH as coenzyme. Structural analysis suggests that specificity for NADPH is conferred by two highly conserved basic residues in the N-terminal part of the peptide chain, whereas specificity for NAD correlates with the presence of an Asp adjacent to the position of the distal basic residue in NADP-dependent enzymes. We carried out site-directed mutagenesis of the two basic residues: Lys-15 and Arg-38, as well as of Ala-37 of human monomeric carbonyl reductase in order to investigate their contribution to coenzyme binding and specificity. Substitution of Lys-15 or Arg-38 by Gln and, even more pronounced Asp decreased the catalytic efficiency (k(cat)/K(m,NADPH)) by more than three orders of magnitude. Similarly, substitution of Asp for Ala-37 decreased k(cat)/K(m,NADPH) 1000-fold but had little effect on k(cat)/K(m,NADH). The results demonstrate the importance of basic residues at positions 15 and 38 and the absence of an acidic residue at position 37 for NADPH binding and catalysis.     

Arginine 165/arginine 277 pair in (S)-mandelate dehydrogenase from Pseudomonas putida: role in catalysis and substrate binding

(S)-Mandelate dehydrogenase from Pseudomonas putida belongs to a FMN-dependent enzyme family that oxidizes (S)-alpha-hydroxyacids. Active site structures of three homologous enzymes, including MDH, show the presence of two conserved arginine residues in close juxtaposition (Arg165 and Arg277 in MDH). Arg277 has an important catalytic role; it stabilizes both the ground and transition states through its positive charge as well as a hydrogen bond [Lehoux, I. E., and Mitra, B. (2000) Biochemistry 39, 10055-10065]. In this study, we examined the role of Arg165 and the overall importance of the Arg165/Arg277 pair. Single mutants at Arg165 as well as double mutants at Arg165 and Arg277 were characterized. Our results show that Arg165 has a role similar to, but less critical than, that of Arg277. It stabilizes the transition state through its positive charge and the ground state through a charge-independent interaction, most likely, a hydrogen bond. Though the k(cat)s for the charge-conserved mutants, R165K and R277K, were only 3-5-fold lower than those of wild-type MDH (wtMDH), the k(cat) for R165K/R277K was approximately 350-fold lower. Thus, at least one arginine residue is required for the optimal substrate orientation and catalysis. Stopped-flow studies show that the FMN reduction step is completely rate-limiting for both wtMDH and the arginine mutants, with the possible exception of R165E. Substrate isotope effects indicate that the carbon-hydrogen bond-breaking step is only partially rate-limiting for wtMDH but fully rate-limiting for the mutants. pH profiles of R165M conclusively show that the pK(a) of 9.3 in free wtMDH does not belong to Arg165.     

Novel insights into the biotin carboxylase domain reactions of pyruvate carboxylase from Rhizobium etli

The catalytic mechanism of the MgATP-dependent carboxylation of biotin in the biotin carboxylase domain of pyruvate carboxylase from R. etli (RePC) is common to the biotin-dependent carboxylases. The current site-directed mutagenesis study has clarified the catalytic functions of several residues proposed to be pivotal in MgATP-binding and cleavage (Glu218 and Lys245), HCO(3)(-) deprotonation (Glu305 and Arg301), and biotin enolization (Arg353). The E218A mutant was inactive for any reaction involving the BC domain and the E218Q mutant exhibited a 75-fold decrease in k(cat) for both pyruvate carboxylation and the full reverse reaction. The E305A mutant also showed a 75- and 80-fold decrease in k(cat) for both pyruvate carboxylation and the full reverse reaction, respectively. While Glu305 appears to be the active site base which deprotonates HCO(3)(-), Lys245, Glu218, and Arg301 are proposed to contribute to catalysis through substrate binding interactions. The reactions of the biotin carboxylase and carboxyl transferase domains were uncoupled in the R353M-catalyzed reactions, indicating that Arg353 may not only facilitate the formation of the biotin enolate but also assist in coordinating catalysis between the two spatially distinct active sites. The 2.5- and 4-fold increase in k(cat) for the full reverse reaction with the R353K and R353M mutants, respectively, suggests that mutation of Arg353 allows carboxybiotin increased access to the biotin carboxylase domain active site. The proposed chemical mechanism is initiated by the deprotonation of HCO(3)(-) by Glu305 and concurrent nucleophilic attack on the γ-phosphate of MgATP. The trianionic carboxyphosphate intermediate formed reversibly decomposes in the active site to CO(2) and PO(4)(3-). PO(4)(3-) then acts as the base to deprotonate the tethered biotin at the N(1)-position. Stabilized by interactions between the ureido oxygen and Arg353, the biotin-enolate reacts with CO(2) to give carboxybiotin. The formation of a distinct salt bridge between Arg353 and Glu248 is proposed to aid in partially precluding carboxybiotin from reentering the biotin carboxylase active site, thus preventing its premature decarboxylation prior to the binding of a carboxyl acceptor in the carboxyl transferase domain.     

Identification of critical residues of choline kinase A2 from Caenorhabditis elegans

Choline kinase catalyzes the phosphorylation of choline by ATP, the first committed step in the CDP-choline pathway for phosphatidylcholine biosynthesis. To begin to elucidate the mechanism of catalysis by this enzyme, choline kinase A-2 from Caenorhabditis elegans was analyzed by systematic mutagenesis of highly conserved residues followed by analysis of kinetic and structural parameters. Specifically, mutants were analyzed with respect to K(m) and k(cat) values for each substrate and Mg(2+), inhibitory constants for Mg(2+) and Ca(2+), secondary structure as monitored by circular dichroism, and sensitivity to unfolding in guanidinium hydrochloride. The most severe impairment of catalysis occurred with the modification of Asp-255 and Asn-260, which are located in the conserved Brenner's phosphotransferase motif, and Asp-301 and Glu-303, in the signature choline kinase motif. For example, mutation of Asp-255 or Asp-301 to Ala eliminated detectable catalytic activity, and mutation of Asn-260 and Glu-303 to Ala decreased k(cat) by 300- and 10-fold, respectively. Additionally, the K(m) for Mg(2+) for mutants N260A and E303A was approximately 30-fold higher than that of wild type. Several other residues (Ser-86, Arg-111, Glu-125, and Trp-387) were identified as being important: Catalytic efficiencies (k(cat)/K(m)) for the enzymes in which these residues were mutated to Ala were reduced to 2-25% of wild type. The high degree of structural similarity among choline kinase A-2, aminoglycoside phosphotransferases, and protein kinases, together with the results from this mutational analysis, indicates it is likely that these conserved residues are located at the catalytic core of choline kinase.     

Molecular mechanisms of improvement of hydrolytic antibody 6D9 by site-directed mutagenesis

We performed a series of site-directed mutagenesis experiments of catalytic antibody, 6D9, which hydrolyzes a prodrug of chloramphenicol, based on our previous directed evolution study [Takahashi et al. (2001) Nat. Biotechnol. 19, 563-567]. Since we previously found that the variants with a mutation of Ser(L27e)Tyr afforded a one order of magnitude increase in catalytic rate, we created a site-directed mutant containing this mutation. The resulting mutant, 6D9-Ser(L27e)Tyr, had 6.5-fold higher k(cat)/k(uncat) and 9.8-fold higher k(cat)/K(m) than wild-type 6D9. We also created 6D9-Thr(L27a)Pro, since this mutation occurred frequently in the previous directed evolution, and it had 2.1-fold higher k(cat)/k(uncat) and k(cat)/K(m) than 6D9. Kinetic and computational analyses suggest that Tyr at L27e contributes to transition-state stabilization, while Pro at L27a does not interact with the transition-state structure directly, but obviously contributes to enhanced catalytic activity. Including double mutants that combined favourable substitutions, we created seven site-directed mutants. However, none of them had higher catalytic activities than some of highly improved variants obtained in the previous directed evolution. The present study gives direct evidence that not only a specific amino acid residue which obviously contributes to transition-state stabilization, but also a group of amino acid residues working in concert is important for efficient catalysis of a given transformation.     

Chemical Rescue and Inhibition Studies to Determine the Role of Arg301 in Phosphite Dehydrogenase

Phosphite dehydrogenase (PTDH) catalyzes the NAD⁺-dependent oxidation of phosphite to phosphate. This reaction requires the deprotonation of a water nucleophile for attack on phosphite. A crystal structure was recently solved that identified Arg301 as a potential base given its proximity and orientation to the substrates and a water molecule within the active site. Mutants of this residue showed its importance for efficient catalysis, with about a 100-fold loss in k _cat and substantially increased K _m,phosphite for the Ala mutant (R301A). The 2.35 Å resolution crystal structure of the R301A mutant with NAD⁺ bound shows that removal of the guanidine group renders the active site solvent exposed, suggesting the possibility of chemical rescue of activity. We show that the catalytic activity of this mutant is restored to near wild-type levels by the addition of exogenous guanidinium analogues; Brønsted analysis of the rates of chemical rescue suggests that protonation of the rescue reagent is complete in the transition state of the rate-limiting step. Kinetic isotope effects on the reaction in the presence of rescue agents show that hydride transfer remains at least partially rate-limiting, and inhibition experiments show that K _i of sulfite with R301A is ∼400-fold increased compared to the parent enzyme, similar to the increase in K _m for phosphite in this mutant. The results of our experiments indicate that Arg301 plays an important role in phosphite binding as well as catalysis, but that it is not likely to act as an active site base.     

Effects on general acid catalysis from mutations of the invariant tryptophan and arginine residues in the protein tyrosine phosphatase from Yersinia

General acid catalysis in protein tyrosine phosphatases (PTPases) is accomplished by a conserved Asp residue, which is brought into position for catalysis by movement of a flexible loop that occurs upon binding of substrate. With the PTPase from Yersinia, we have examined the effect on general acid catalysis caused by mutations to two conserved residues that are integral to this conformation change. Residue Trp354 is at a hinge of the loop, and Arg409 forms hydrogen bonding and ionic interactions with the phosphoryl group of substrates. Trp354 was mutated to Phe and to Ala, and residue Arg409 was mutated to Lys and to Ala. The four mutant enzymes were studied using steady state kinetics and heavy-atom isotope effects with the substrate p-nitrophenyl phosphate. The data indicate that mutation of the hinge residue Trp354 to Ala completely disables general acid catalysis. In the Phe mutant, general acid catalysis is partially effective, but the proton is only partially transferred in the transition state, in contrast to the native enzyme where proton transfer to the leaving group is virtually complete. Mutation of Arg409 to Lys has a minimal effect on the K(m), while this parameter is increased 30-fold in the Ala mutant. The k(cat) values for R409K and for R409A are about 4 orders of magnitude lower than that for the native enzyme. General acid catalysis is rendered inoperative by the Lys mutation, but partial proton transfer during catalysis still occurs in the Ala mutant. Structural explanations for the differential effects of these mutations on movement of the flexible loop that enables general acid catalysis are presented.     

Putative reaction mechanism of heterologously expressed octopine dehydrogenase from the great scallop, Pecten maximus (L)

cDNA for octopine dehydrogenase (ODH) from the adductor muscle of the great scallop, Pecten maximus, was cloned using 5'- and 3'-RACE. The cDNA comprises an ORF of 1197 nucleotides and the deduced amino acid sequence encodes a protein of 399 amino acids. ODH was heterologously expressed in Escherichia coli with a C-terminal penta His-tag. ODH-5His was purified to homogeneity using metal-chelate affinity chromatography and Sephadex G-100 gel filtration. Recombinant ODH had kinetic properties similar to those of wild-type ODH isolated from the scallop's adductor muscle. Site-directed mutagenesis was used to elucidate the involvement of several amino acid residues for the reaction catalyzed by ODH. Cys148, which is conserved in all opine dehydrogenases known to date, was converted to serine or alanine, showing that this residue is not intrinsically important for catalysis. His212, Arg324 and Asp329, which are also conserved in all known opine dehydrogenase sequences, were subjected to site-directed mutagenesis. Modification of these residues revealed their importance for the catalytic activity of the enzyme. Conversion of each of these residues to alanine resulted in strong increases in K(m) and decreases in k(cat) values for pyruvate and L-arginine, but had little effect on the K(m) and k(cat) values for NADH. Assuming a similar structure for ODH compared with the only available structure of a bacterial opine dehydrogenase, these three amino acids may function as a catalytic triad in ODH similar to that found in lactate dehydrogenase or malate dehydrogenase. The carboxyl group of pyruvate is then stabilized by Arg324. In addition to orienting the substrate, His212 will act as an acid-base catalyst by donating a proton to the carbonyl group of pyruvate. The acidity of this histidine is further increased by the proximity of Asp329.     

Relaxing the nicotinamide cofactor specificity of phosphite dehydrogenase by rational design

Homology modeling was used to identify two particular residues, Glu175 and Ala176, in Pseudomonas stutzeri phosphite dehydrogenase (PTDH) as the principal determinants of nicotinamide cofactor (NAD(+) and NADP(+)) specificity. Replacement of these two residues by site-directed mutagenesis with Ala175 and Arg176 both separately and in combination resulted in PTDH mutants with relaxed cofactor specificity. All three mutants exhibited significantly better catalytic efficiency for both cofactors, with the best kinetic parameters displayed by the double mutant, which had a 3.6-fold higher catalytic efficiency for NAD(+) and a 1000-fold higher efficiency for NADP(+). The cofactor specificity was changed from 100-fold in favor of NAD(+) for the wild-type enzyme to 3-fold in favor of NADP(+) for the double mutant. Isoelectric focusing of the proteins in a nondenaturing gel showed that the replacement with more basic residues indeed changed the effective pI of the protein. HPLC analysis of the enzymatic products of the double mutant verified that the reaction proceeded to completion using either substrate and produced only the corresponding reduced cofactor and phosphate. Thermal inactivation studies showed that the double mutant was protected from thermal inactivation by both cofactors, while the wild-type enzyme was protected by only NAD(+). The combined results provide clear evidence that Glu175 and Ala176 are both critical for nicotinamide cofactor specificity. The rationally designed double mutant might be useful for the development of an efficient in vitro NAD(P)H regeneration system for reductive biocatalysis.     

Molecular determinants of the cofactor specificity of ribitol dehydrogenase, a short-chain dehydrogenase/reductase

Ribitol dehydrogenase from Zymomonas mobilis (ZmRDH) catalyzes the conversion of ribitol to d-ribulose and concomitantly reduces NAD(P)(+) to NAD(P)H. A systematic approach involving an initial sequence alignment-based residue screening, followed by a homology model-based screening and site-directed mutagenesis of the screened residues, was used to study the molecular determinants of the cofactor specificity of ZmRDH. A homologous conserved amino acid, Ser156, in the substrate-binding pocket of the wild-type ZmRDH was identified as an important residue affecting the cofactor specificity of ZmRDH. Further insights into the function of the Ser156 residue were obtained by substituting it with other hydrophobic nonpolar or polar amino acids. Substituting Ser156 with the negatively charged amino acids (Asp and Glu) altered the cofactor specificity of ZmRDH toward NAD(+) (S156D, [k(cat)/K(m)(,NAD)]/[k(cat)/K(m)(,NADP)] = 10.9, where K(m)(,NAD) is the K(m) for NAD(+) and K(m)(,NADP) is the K(m) for NADP(+)). In contrast, the mutants containing positively charged amino acids (His, Lys, or Arg) at position 156 showed a higher efficiency with NADP(+) as the cofactor (S156H, [k(cat)/K(m)(,NAD)]/[k(cat)/K(m)(,NADP)] = 0.11). These data, in addition to those of molecular dynamics and isothermal titration calorimetry studies, suggest that the cofactor specificity of ZmRDH can be modulated by manipulating the amino acid residue at position 156.     

Alanine scanning mutagenesis of the testosterone binding site of rat 3 alpha-hydroxysteroid dehydrogenase demonstrates contact residues influence the rate-determining step

Aldo-keto reductase (AKR1C) isoforms can regulate ligand access to nuclear receptors by acting as hydroxysteroid dehydrogenases. The principles that govern steroid hormone binding and steroid turnover by these enzymes were analyzed using rat 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD, AKR1C9) as the protein model. Systematic alanine scanning mutagenesis was performed on the substrate-binding pocket as defined by the crystal structure of the 3alpha-HSD.NADP(+).testosterone ternary complex. T24, L54, F118, F129, T226, W227, N306, and Y310 were individually mutated to alanine, while catalytic residues Y55 and H117 were unaltered. The effects of these mutations on the ordered bi-bi mechanism were examined. No mutations changed the affinity for NADPH by more than 2-3-fold. Fluorescence titrations of the energy transfer band of the E.NADPH complex with competitive inhibitors testosterone and progesterone showed that the largest effect was a 23-fold decrease in the affinity for progesterone in the W227A mutant. By contrast, changes in the K(d) for testosterone were negligible. Examination of the k(cat)/K(m) data for these mutants indicated that, irrespective of steroid substrate, the bimolecular rate constant was more adversely affected when alanine replaced an aromatic hydrophobic residue. By far, the greatest effects were on k(cat) (decreases of more than 2 log units), suggesting that the rate-determining step was either altered or slowed significantly. Single- and multiple-turnover experiments for androsterone oxidation showed that while the wild-type enzyme demonstrated a k(lim) and burst kinetics consistent with slow product release, the W227A and F118A mutants eliminated this kinetic profile. Instead, single- and multiple-turnover experiments gave k(lim) and k(max) values identical with k(cat) values, respectively, indicating that chemistry was now rate-limiting overall. Thus, conserved residues within the steroid-binding pocket affect k(cat) more than K(d) by influencing the rate-determining step of steroid oxidation. These findings support the concept of enzyme catalysis in which the correct positioning of reactants is essential; otherwise, k(cat) will be limited by the chemical event.     

Contribution of active-site residues to the function of onconase, a ribonuclease with antitumoral activity

Onconase (ONC), a homologue of ribonuclease A (RNase A), is in clinical trials for the treatment of cancer. ONC possesses a conserved active-site catalytic triad, which is composed of His10, Lys31, and His97. The three-dimensional structure of ONC suggests that two additional residues, Lys9 and an N-terminal lactam formed from a glutamine residue (Pca1), could also contribute to catalysis. To determine the role of Pca1, Lys9, and Lys31 in the function of ONC, site-directed mutagenesis was used to replace each with alanine. Values of k(cat)/K(M) for the variants were determined with a novel fluorogenic substrate, which was designed to match the nucleobase specificity of ONC and gives the highest known k(cat)/K(M) value for the enzyme. The K9A and K31A variants display 10(3)-fold lower k(cat)/K(M) values than the wild-type enzyme, and a K9A/K31A double variant suffers a >10(4)-fold decrease in catalytic activity. In addition, replacing Lys9 or Lys31 eliminates the antitumoral activity of ONC. The side chains of Pca1 and Lys9 form a hydrogen bond in crystalline ONC. Replacing Pca1 with an alanine residue lowers the catalytic activity of ONC by 20-fold. Yet, replacing Pca1 in the K9A variant enzyme does not further reduce catalytic activity, revealing that the function of the N-terminal pyroglutamate residue is to secure Lys9. The thermodynamic cycle derived from k(cat)/K(M) values indicates that the Pca1...Lys9 hydrogen bond contributes 2.0 kcal/mol to the stabilization of the rate-limiting transition state during catalysis. Finally, binding isotherms with a substrate analogue indicate that Lys9 and Lys31 contribute little to substrate binding and that the low intrinsic catalytic activity of ONC originates largely from the low affinity of the enzyme for its substrate. These findings could assist the further development of ONC as a cancer chemotherapeutic.     

Identification of critical steps governing the two-component alkanesulfonate monooxygenase catalytic mechanism

The alkanesulfonate monooxygenase enzyme (SsuD) catalyzes the oxygenolytic cleavage of a carbon-sulfur bond from sulfonated substrates. A mechanism involving acid-base catalysis has been proposed for the desulfonation mechanism by SsuD. In the proposed mechanism, base catalysis is involved in abstracting a proton from the alkane peroxyflavin intermediate, while acid catalysis is needed for the protonation of the FMNO(-) intermediate. The pH profiles of k(cat) indicate that catalysis by SsuD requires a group with a pK(a) of 6.6 ± 0.2 to be deprotonated and a second group with a pK(a) of 9.5 ± 0.1 to be protonated. The upper pK(a) value was not present in the pH profiles of k(cat)/K(m). Several conserved amino acid residues (His228, His11, His333, Cys54, and Arg226) have been identified as having potential catalytic importance due to the similar spatial arrangements with close structural and functional relatives of SsuD. Substitutions to these amino acid residues were generated, and the pH dependencies were evaluated and compared to wild-type SsuD. Although a histidine residue was previously proposed to be the active site base, the His variants possessed similar steady-state kinetic parameters as wild-type SsuD. Interestingly, R226A and R226K SsuD variants possessed undetectable activity, and there was no detectable formation of the C4a-(hydro)peroxyflavin intermediate for the Arg226 SsuD variants. Guanidinium rescue with the R226A SsuD variant resulted in the recovery of 1.5% of the wild-type SsuD k(cat) value. These results implicate Arg226 playing a critical role in catalysis and provide essential insights into the mechanistic steps that guide the SsuD desulfonation process.     

The roles of Tyr(91) and Lys(162) in general acid-base catalysis in the pigeon NADP+-dependent malic enzyme

The role of general acid-base catalysis in the enzymatic mechanism of NADP+-dependent malic enzyme was examined by detailed steady-state kinetic studies through site-directed mutagenesis of the Tyr(91) and Lys(162) residues in the putative catalytic site of the enzyme. Y91F and K162A mutants showed approx. 200- and 27000-fold decreases in k(cat) values respectively, which could be partially recovered with ammonium chloride. Neither mutant had an effect on the partial dehydrogenase activity of the enzyme. However, both Y91F and K162A mutants caused decreases in the k(cat) values of the partial decarboxylase activity of the enzyme by approx. 14- and 3250-fold respectively. The pH-log(k(cat)) profile of K162A was found to be different from the bell-shaped profile pattern of wild-type enzyme as it lacked a basic pK(a) value. Oxaloacetate, in the presence of NADPH, can be converted by malic enzyme into L-malate by reduction and into enolpyruvate by decarboxylation activities. Compared with wild-type, the K162A mutant preferred oxaloacetate reduction to decarboxylation. These results are consistent with the function of Lys(162) as a general acid that protonates the C-3 of enolpyruvate to form pyruvate. The Tyr(91) residue could form a hydrogen bond with Lys(162) to act as a catalytic dyad that contributes a proton to complete the enol-keto tautomerization.     

Conserved and nonconserved residues in the substrate binding site of 7,8-diaminopelargonic acid synthase from Escherichia coli are essential for catalysis

The vitamin B(6)-dependent enzyme 7,8-diaminopelargonic acid (DAPA) synthase catalyzes the antepenultimate step in the synthesis of biotin, the transfer of the alpha-amino group of S-adenosyl-l-methionine (SAM) to 7-keto-8-aminopelargonic acid (KAPA) to form DAPA. The Y17F, Y144F, and D147N mutations in the active site were constructed independently. The k(max)/K(m)(app) values for the half-reaction with DAPA of the Y17F and Y144F mutants are reduced by 1300- and 2900-fold, respectively, compared to the WT enzyme. Crystallographic analyses of these mutants do not show significant changes in the structure of the active site. The kinetic deficiencies, together with a structural model of the enzyme-PLP/DAPA Michaelis complex, point to a role of these two residues in recognition of the DAPA/KAPA substrates and in catalysis. The k(max)/K(m)(app) values for the half-reaction with SAM are similar to that of the WT enzyme, showing that the two tyrosine residues are not involved in this half-reaction. Mutations of the conserved Arg253 uniquely affect the SAM kinetics, thus establishing this position as part of the SAM binding site. The D147N mutant is catalytically inactive in both half-reactions. The structure of this mutant exhibits significant changes in the active site, indicating that this residue plays an important structural role. Of the four residues examined, only Tyr144 and Arg253 are strictly conserved in the available amino acid sequences of DAPA synthases. This enzyme thus provides an illustrative example that active site residues essential for catalysis are not necessarily conserved, i.e., that during evolution alternative solutions for efficient catalysis by the same enzyme arose. Decarboxylated SAM [S-adenosyl-(5')-3-methylthiopropylamine] reacts nearly as well as SAM and cannot be eliminated as a putative in vivo amino donor.     

人双专一性磷酸酶活性位点Cys^124附近精氨酸突变及功能

为研究人双专一性磷酸酶活性位点Cys12 4 附近 3个带正电的精氨酸对酶催化功能的影响 ,用QuikChange定点突变方法获得 6个突变体 :R12 5L、R130 L、R130 K、R130 L/S131A、R158K和R158L。将含突变基因的重组质粒转化大肠杆菌菌株BL2 1(DE3) ,经IPTG诱导表达获得的目的蛋白质均以可溶形式存在。通过镍离子亲和层析纯化得到纯度大于 90 %的蛋白质。对人痘苗病毒H1相关磷酸酶 (VHR)及其突变体进行稳态动力学参数和竞争性抑制常数Ki 的测定 ,结果显示上述Arg130 和Arg158突变体的kcat/Km 值都较野生型有大幅度下降 ,而Ki 值有明显上升 ,表明 130和 15 8位的精氨酸是VHR活性所必需 ,而且可能与底物上带负电的磷酸基团结合有关。另外 ,单突变体R130 L和双突变体R130 L/S131A之间的kcat值相差很小 ,提示Arg130 单点突变后可能破坏了Ser131与Cys12 4 间的氢键。再者 ,R12 5L、R130 L和R158L突变体都降低了砷酸盐结合亲和性 ,暗示这 3个精氨酸残基侧链上的正电荷可能有助于底物与酶的结合。     

A structure-function study of a proton transport pathway in the gamma-class carbonic anhydrase from Methanosarcina thermophila

Four glutamate residues in the prototypic gamma-class carbonic anhydrase from Methanosarcina thermophila (Cam) were characterized by site-directed mutagenesis and chemical rescue studies. Alanine substitution indicated that an external loop residue, Glu 84, and an internal active site residue, Glu 62, are both important for CO(2) hydration activity. Two other external loop residues, Glu 88 and Glu 89, are less important for enzyme function. The two E84D and -H variants exhibited significant activity relative to wild-type activity in pH 7.5 MOPS buffer, suggesting that the original glutamate residue could be substituted with other ionizable residues with similar pK(a) values. The E84A, -C, -K, -Q, -S, and -Y variants exhibited large decreases in k(cat) values in pH 7.5 MOPS buffer, but only exhibited small changes in k(cat)/K(m). These same six variants were all chemically rescued by pH 7.5 imidazole buffer, with 23-46-fold increases in the apparent k(cat). These results are consistent with Glu 84 functioning as a proton shuttle residue. The E62D variant exhibited a 3-fold decrease in k(cat) and a 2-fold decrease in k(cat)/K(m) relative to those of the wild type in pH 7.5 MOPS buffer, while other substitutions (E62A, -C, -H, -Q, -T, and -Y) resulted in much larger decreases in both k(cat) and k(cat)/K(m). Imidazole did not significantly increase the k(cat) values and slightly decreased the k(cat)/K(m) values of most of the Glu 62 variants. These results indicate a primary preference for a carboxylate group at position 62, and support a proposed catalytic role for residue Glu 62 in the CO(2) hydration step, but do not definitively establish its role in the proton transport step.