Mechanistic roles of Thr134, Tyr160, and Lys 164 in the reaction catalyzed by dTDP-glucose 4,6-dehydratase

Escherichia coli dTDP-glucose 4,6-dehydratase and UDP-galactose 4-epimerase are members of the short-chain dehydrogenase/reductase SDR family. A highly conserved triad consisting of Ser/Thr, Tyr, and Lys is present in the active sites of these enzymes as well in other SDR proteins. Ser124, Tyr149, and Lys153 in the active site of UDP-galactose 4-epimerase are located in similar positions as the corresponding Thr134, Tyr160, and Lys164, in the active site of dTDP-glucose 4,6-dehydratase. The role of these residues in the first hydride transfer step of the dTDP-glucose 4,6-dehydratase mechanism has been studied by mutagenesis and steady-state kinetic analysis. In all mutants except T134S, the k(cat) values are more than 2 orders of magnitude lower than of wild-type enzyme. The substrate analogue, dTDP-xylose, was used to investigate the effects of the mutations on rate of the first hydride transfer step. The first step becomes significantly rate limiting upon mutation of Tyr160 to Phe and only partly rate limiting in the reaction catalyzed by K164M and T134A dehydratases. The pH dependence of k(cat), the steady-state NADH level, and the fraction of NADH formed with saturating dTDP-xylose show shifts in the pK(a) assigned to Tyr160 to more basic values by mutation of Lys164 and Thr134. The pK(a) of Tyr160, as determined by the pH dependence of NADH formation by dTDP-xylose, is 6.41. Lys164 and Thr134 are believed to play important roles in the stabilization of the anion of Tyr160 in a fashion similar to the roles of the corresponding residues in UDP-galactose 4-epimerase, which facilitate the ionization of Tyr149 in that enzyme [Liu, Y., et al. (1997) Biochemistry 35, 10675--10684]. Tyr160 is presumably the base for the first hydride transfer step, while Thr134 may relay a proton from the sugar to Tyr160.     

Overexpression of the recombinant Enterobacter cloacae P99 AmpC beta-lactamase and its mutants based on a phi105 prophage system in Bacillus subtilis

AmpC beta-lactamase is a bacterial enzyme with great clinical impact as it mediates beta-lactam antibiotic resistance in many Gram-negative bacteria. To facilitate the structure-function relationship studies on this clinically important enzyme, we developed new strategies for production of recombinant Enterobacter cloacae P99 AmpC beta-lactamase in Bacillus subtilis. With the utilization of a special thermo-inducible phi105 phage system, functionally active AmpC beta-lactamase was expressed in B. subtilis, either in an extracellular native form or an intracellular N-terminal (His)(6)-tagged form. A higher expression level was achieved when expressing the enzyme as the intracellular (His)(6)-tagged protein rather than as the extracellular native protein. In addition, from the approach of producing intracellular tagged protein, highly pure (>95%) (His)(6)-tagged beta-lactamase wild-type and mutants (Y150C and K315C) were obtained after a one-step nickel affinity chromatography with a yield of 28.5, 66, and 0.85 mg/L of culture, respectively. Furthermore, the Y150C and K315C mutants were characterized so as to investigate the roles of the conserved residues, Tyr150 and Lys315, in the AmpC beta-lactamase. Severe impairment in hydrolytic abilities and restored secondary structures of the Y150C and K315C mutants suggested the major contribution of these two residues in the catalytic reaction rather than the structural framework in the AmpC enzyme.     

Pyridoxal 5'-phosphate as a 31P reporter observing functional changes in the active site of Escherichia coli maltodextrin phosphorylase after site-directed mutagenesis.

Changes in the active site of Escherichia coli maltodextrin phosphorylase created by substituting residues Lys533, Arg534, Tyr538, and Glu637 were monitored in the absence and presence of arsenate as substrate analogue using pyridoxal-P as 31P NMR reporter. The chemical shift of the cofactor phosphate group of wild-type E. coli phosphorylase is pH dependent with an apparent pK of 5.6 and limiting delta values of 0.71 and 3.6 ppm for the low- and high-pH values, respectively. The apparent pK value of 5.6 indicates that the phosphate group of the cofactor is in hydrogen bond linkage to Lys533. In all mutant enzymes in which the enzymatic activity was significantly reduced, effects on the 31P chemical shift pattern of pyridoxal-P were observed. The K533S, R534Q, E637D, and E637Q mutant enzymes show 0.6, 0.01, 0.2, or 0.1% residual activity, and the apparent pK values of the cofactor phosphate transition of E637D and E637Q mutant enzymes are altered. The Y538F mutant enzyme is a remarkable exception, displaying 12% activity and an environment of the cofactor quite similar to that in wild-type enzyme. This finding suggests that Tyr538, although involved in substrate binding and specificity, is not functionally essential. One crucial aspect of catalysis is the close contact of the phosphates of pyridoxal-P and of substrate rendered by a cluster of positively charged amino acids, Lys533, Lys539, and Arg534. The similar apparent pK values of wild-type and K533S mutant phosphorylase suggest that the cofactor phosphate and the hydroxyl group of Ser533 are linked by a hydrogen bond.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of the extended-spectrum class C beta-lactamase of Enterobacter cloacae GC1, a natural mutant with a tandem tripeptide insertion.

A class C beta-lactamase from a clinical isolate of Enterobacter cloacae strain GC1 with improved hydrolytic activity for oxyimino beta-lactam antibiotics has been analyzed by X-ray crystallography to 1.8 A resolution. Relative to the wild-type P99 beta-lactamase, this natural mutant contains a highly unique tandem repeat Ala211-Val212-Arg213 [Nugaka et al. (1995) J. Biol. Chem. 270, 5729-5735]. The 39.4 kDa chromosomal beta-lactamase crystallizes from poly(ethylene glycol) 8000 in potassium phosphate in space group P2(1)2(1)2 with cell dimensions a = 78.0 A, b = 69.5 A, and c = 63.1 A. The crystal structure was solved by the molecular replacement method, and the model has been refined to an R-factor of 0.20 for all nonzero data from 8 to 1.8 A. Deviations of model bonds and angles from ideal values are 0.008 A and 1.4 degrees, respectively. Overlay of alpha-carbon atoms in the GC1 and P99 beta-lactamases results in an rms deviation of 0.6 A. Largest deviations occur in a loop containing Gln120 and in the Omega loop region (200-218) where the three residues 213-215 are disordered. Possibly as a result of this disorder, the width of the opening to the substrate binding cavity, as measured from the 318-324 beta-strand to two loops containing Gln120 and Tyr150 on the other side, is 0.6-1.4 A wider than in P99. It is suggested that conformational flexibility in the expanded Omega loop, and its influence on adjacent protein structure, may facilitate hydrolysis of oxyimino beta-lactams by making the acyl intermediate more open to attack by water. Nevertheless, backbone atoms in core catalytic site residues Ser64, Lys67, Tyr150, Asn152, Lys318, and Ser321 deviate only 0.4 A (rmsd) from atoms in P99. A rotation of a potential catalytic base, Tyr150, relative to P99 at pH 8, is consistent with the requirement for a lower than normal pK(a) for this residue.     

Electrostatic interactions in ubiquitin: stabilization of carboxylates by lysine amino groups

To explore electrostatic interactions in ubiquitin, pK(a) values have been determined by NMR for all 12 carboxyl groups in wild-type ubiquitin and in variants where single lysines have been replaced by neutral residues. Aspartate pK(a) values in ubiquitin range from 3.1 to 3.8 and are generally less than model compound values. Most aspartate pK(a) values are within 0.2 pH unit of those predicted with a simple Tanford-Kirkwood model. Glutamate pK(a) values range from 3.8 to 4.5, close to model compound values and differing by 0.1-0.8 pH unit from calculated values. To determine the role of positive charges in modulating carboxyl pK(a) values, we mutated lysines at positions 11, 29, and 33 to glutamine and threonine. NMR studies with these six single-site mutants reveal significant interactions of Lys 11 and Lys 29 with Glu 34 and Asp 21, respectively: pK(a) values for Glu 34 and Asp 21 increase by approximately 0.5-0.8 pH unit, similar to predicted values, when the lysines are replaced by neutral residues. In contrast, the predicted interaction between Lys 33 and Glu 34 is not observed experimentally. In some instances, substitution of lysine by glutamine and threonine did not lead to the same changes in carboxyl pK(a) values. These may reflect new short-range interactions between the mutated residues and the carboxyl groups. Carboxyl pK(a) shifts > 0.5 pH unit result from mutations at groups that are <5 A from the carboxyl group. No interactions are observed at >10 A.     

Participation of the Lys313-Ile333 sequence of the purinergic P2X4 receptor in agonist binding and transduction of signals to the channel gate

To study the roles of the Lys(313)-Ile(333) ectodomain sequence of the rat P2X(4) receptor in ATP binding and transduction of signals to the channel gate, the conserved Lys(313), Tyr(315), Gly(316), Ike(317), Arg(318), Asp(320), Val(323), Lys(329), Phe(330), and Ile(333) residues were mutated. Current recordings were done on lifted cells and ATP was applied using an ultrafast solution-switching system. The rates of wild type channel opening and closing in the presence of ATP, but not the rate of washout-induced closing, were dependent on agonist concentration. All mutants other than I317A were expressed in the plasma membrane at comparable levels. The majority of mutants showed significant changes in the peak amplitude of responses and the EC(50) values for ATP. When stimulated with the supramaximal (1.4 mm) ATP concentration, mutants also differed in the kinetics of their activation, deactivation, and/or desensitization. The results suggest a critical role of the Lys(313) residue in receptor function other than coordination of the phosphate group of ATP and possible contribution of the Tyr(315) residue to the agonist binding module. The pattern of changes of receptor function by mutation of other residues was consistent with the operation of the Gly(316)-Ile(333) sequence as a signal transduction module between the ligand binding domain and the channel gate in the second transmembrane domain.     

Insensitivity of perturbed carboxyl pK(a) values in the ovomucoid third domain to charge replacement at a neighboring residue

A number of carboxyl groups in turkey ovomucoid third domain (OMTKY3) have low pK(a) values. A previous study suggested that neighboring amino groups were primarily responsible for the low carboxyl pK(a) values. However, the expected elevation in pK(a) values for these amino groups was not observed. In the present study, site-directed mutagenesis is used to investigate the origins of perturbed carboxyl pK(a) values in OMTKY3. Electrostatic calculations suggest that Lys 34 has large effects, 0.4-0.6 unit, on Asp 7, Glu 10, and Glu 19 which are 5-11 A away from Lys 34. Two-dimensional (1)H NMR techniques were used to determine pK(a) values of the acidic residues in OMTKY3 mutants in which Lys 34 has been replaced with threonine and glutamine. Surprisingly, the pK(a) values in the mutants are very close to those of the wild-type protein. The insensitivity of the acidic residues to replacement of Lys 34 suggests that long-range electrostatic interactions play less of a role in perturbing carboxyl pK(a) values than originally thought. We hypothesize that hydrogen bonds play a key role in perturbing some of the carboxyl ionization equilibria in OMTKY3.     

The roles of residues Tyr150, Glu272, and His314 in class C β-lactamases

Serine β-lactamases contribute widely to the β-lactam resistance phenomena. Unfortunately, the intimate details of their catalytic mechanism remain elusive and subject to some controversy even though many “natural” and “artificial” mutants of these different enzymes have been isolated. This paper is essentially focused on class C β-lactamases, which contain a Tyr (Tyr150) as the first residue of the second conserved element, in contrast to their class A counterparts, in which a Ser is found in the corresponding position. We have modified this Tyr residue by site-directed mutagenesis. On the basis of the three-dimensional structure of the Enterobacter cloacae P99 enzyme, it seemed that residues Glu272 and His314 might also be important. They were similarly substituted. The modified enzymes were isolated and their catalytic properties determined. Our results indicated that His314 was not required for catalysis and that Glu272 did not play an important role in acylation but was involved to a small extent in the deacylation process. Conversely, Tyr150 was confirmed to be central for catalysis, at least with the best substrates. On the basis of a comparison of data obtained for several class C enzyme mutants and in agreement with recent structural data, we propose that the phenolate anion of Tyr150, in conjunction with the alkyl ammonium of Lys315, acts as the general base responsible for the activation of the active-site Ser64 during the acylation step and for the subsequent activation of a water molecule in the deacylation process. The evolution of the important superfamily of penicillin-recognizing enzymes is further discussed in the light of this proposed mechanism. © 1996 Wiley-Liss, Inc.     

Rapid Calculation of Protein pK(a) Values Using Rosetta

We developed a Rosetta-based Monte Carlo method to calculate the pK(a) values of protein residues that commonly exhibit variable protonation states (Asp, Glu, Lys, His, and Tyr). We tested the technique by calculating pK(a) values for 264 residues from 34 proteins. The standard Rosetta score function, which is independent of any environmental conditions, failed to capture pK(a) shifts. After incorporating a Coulomb electrostatic potential and optimizing the solvation reference energies for pK(a) calculations, we employed a method that allowed side-chain flexibility and achieved a root mean-square deviation (RMSD) of 0.83 from experimental values (0.68 after discounting 11 predictions with an error over 2 pH units). Additional degrees of side-chain conformational freedom for the proximal residues facilitated the capture of charge-charge interactions in a few cases, resulting in an overall RMSD of 0.85 pH units. The addition of backbone flexibility increased the overall RMSD to 0.93 pH units but improved relative pK(a) predictions for proximal catalytic residues. The method also captures large pK(a) shifts of lysine and some glutamate point mutations in staphylococcal nuclease. Thus, a simple and fast method based on the Rosetta score function and limited conformational sampling produces pK(a) values that will be useful when rapid estimation is essential, such as in docking, design, and folding.     

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.     

Computational and mutational analysis of human glutaredoxin (thioltransferase): probing the molecular basis of the low pKa of cysteine 22 and its role in catalysis

Human glutaredoxin (GRx), also known as thioltransferase, is a 12 kDa thiol-disulfide oxidoreductase that is highly selective for reduction of glutathione-containing mixed disulfides. The apparent pK(a) for the active site Cys22 residue is approximately 3.5. Previously we observed that the catalytic enhancement by glutaredoxin could be ascribed fully to the difference between the pK(a) of its Cys22 thiol moiety and the pK(a) of the product thiol, each acting as a leaving group in the enzymatic and nonenzymatic reactions, respectively [Srinivasan et al. (1997), Biochemistry 36, 3199-3206]. Continuum electrostatic calculations suggest that the low pK(a) of Cys22 results primarily from stabilization of the thiolate anion by a specific ion-pairing with the positively charged Lys19 residue, although hydrogen bonding interactions with Thr21 also appear to contribute. Variants of Lys19 were considered to further assess the predicted role of Lys19 on the pK(a) of Cys22. The variants K19Q and K19L were generated by molecular modeling, and the pK(a) value for Cys22 was calculated for each variant. For K19Q, the predicted Cys22 pK(a) is 7.3, while the predicted value is 8.3 for K19L. The effects of the mutations on the interaction energy between the adducted glutathionyl moiety and GRx were roughly estimated from the van der Waals and electrostatic energies between the glutathionyl moiety and proximal protein residues in a mixed disulfide adduct of GRx and glutathione, i.e., the GRx-SSG intermediate. The values for the K19 mutants differed by only a small amount compared to those for the wild type enzyme intermediate. Together, the computational analysis predicted that the mutant enzymes would have markedly reduced catalytic rates while retaining the glutathionyl specificity displayed by the wild type enzyme. Accordingly, we constructed and characterized the K19L and K19Q mutants of two forms of the GRx enzyme. Each of the mutants retained glutathionyl specificity as predicted and displayed diminution in activity, but the decreases in activity were not to the extent predicted by the theoretical calculations. Changes in the respective Cys22-thiol pK(a) values of the mutant enzymes, as shown by pH profiles for iodoacetamide inactivation of the respective enzymes, clearly revealed that the K19-C22 ion pair cannot fully account for the low pK(a) of the Cys22 thiol. Additional contributions to stabilization of the Cys22 thiolate are likely donated by Thr21 and the N-terminal partial positive charge of the neighboring alpha-helix.     

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.     

Rearrangement of charge-charge interactions in variant ubiquitins as detected by double-mutant cycles and NMR

Previous studies of ubiquitin disclosed numerous charge-charge interactions on the protein's surface. To investigate how neighboring residues influence the strength of these interactions, double-mutant cycles are combined with pK(a) determinations by 2D NMR. More specifically, the environment around the Asp21-Lys29 ion pair has been altered through mutations at position 25, which is an asparagine in mammalian ubiquitin and a positively-charged residue in many other ubiquitin-like proteins. The pK(a) value of Asp21 decreases by 0.4 to 0.7 pH unit when Asn25 is substituted with a positively charged residue, suggesting a new and favorable ion pair interaction between positions 21 and 25. However, analysis of double mutants reveals that the favorable interaction between Asp21 and Lys29 is weakened when position 25 is a positively charged residue. Interestingly, while the pK(a) value of His25 in the N25H variant agrees with model compound values, additional mutants reveal that this agreement is fortuitous, resulting from a balance of favorable and unfavorable interactions; similar results were observed previously for Glu34 in ubiquitin and His8 in staphylococcal nuclease. Ionizable groups may thus have pK(a) values similar to model compound values and yet still be involved in significant interactions with other protein groups. One surprising result of introducing positively charged residues at position 25 is a new interaction between Lys29 and Glu18, an interaction not present in wild-type ubiquitin. This unanticipated result illustrates a key advantage of using NMR to determine pK(a) values for many residues simultaneously in the variant proteins. Overall, the strength of an interaction between two residues at the surface of ubiquitin is sensitive to the identity of neighboring residues. The results also demonstrate that relatively conservative and common point mutations such as substitutions of polar with charged residues and vice versa can have effects on interactions beyond the site of mutation per se.     

Analysis of the catalytic mechanism of pyruvate dehydrogenase kinase

It has been proposed that "Glu238" within the N-box of pyruvate dehydrogenase kinase (PDK) is a base catalyst. The pH dependence of k(cat) of Arabidopsis thaliana PDK indicates that ionizable groups with pK values of 6.2 and 8.4 are necessary for catalysis, and the temperature dependence of these values suggests that the acidic pK is due to a carboxyl- or imidazole-group. The E238 and K241 mutants had elevated K(m,ATP) values. The acidic pK value of the E238A mutant was shifted to 5.5. The H233A, L234H, and L234A mutants had the same pK values as wild-type AtPDK, contrary to the previous proposal of a "Glu-polarizing" His. Instead, we suggest that the conserved Glu, Lys, and Asn residues of the N-box contribute to coordinating Mg2+ in a position critical for formation of the PDK-MgATP-substrate ternary complex.     

Molecular dynamics simulations of class C beta-lactamase from Citrobacter freundii: insights into the base catalyst for acylation

Herein, we present results from molecular dynamics (MD) simulations of the class C beta-lactamase from Citrobacter freundii and its Michaelis complex with aztreonam. Four different configurations of the active site were modeled in aqueous solution, and their relative stability was estimated by means of quantum mechanical energy calculations. For the free enzyme, the energetically most stable configurations present a neutral Lys67 residue or an anionic Tyr150 side chain. Our calculations predict that these two configurations are quite close in terms of free energy, the anionic Tyr150 state being favored by approximately 1 kcal/mol. In contrast, for the noncovalent complex formed between the C. freundii enzyme and aztreonam, the energetic analyses predict that the configuration with the neutral Lys67 residue is much more stable than the anionic Tyr150 one (approximately 20 kcal/mol). Moreover, the MD simulations reveal that the neutral Lys67 state results in a proper enzyme-aztreonam orientation for nucleophilic attack and in a very stable contact between the nucleophilic hydroxyl group of Ser64 and the neutral amino side chain of Lys67. Thus, both the computed free energies and the structural analyses support the assignation of Lys67 as the base catalyst for the acylation step in the native form of the C. freundii enzyme.     

Glutamine 53 is a gatekeeper residue in the FK506 binding protein

The structures of enzymes reflect two tendencies that appear opposed. On one hand, they fold into compact, stable structures; on the other hand, they bind a ligand and catalyze a reaction. To be stable, enzymes fold to maximize favorable interactions, forming a tightly packed hydrophobic core, exposing hydrophilic groups, and optimizing intramolecular hydrogen-bonding. To be functional, enzymes carve out an active site for ligand binding, exposing hydrophobic surface area, clustering like charges, and providing unfulfilled hydrogen bond donors and acceptors. Using AmpC beta-lactamase, an enzyme that is well-characterized structurally and mechanistically, the relationship between enzyme stability and function was investigated by substituting key active-site residues and measuring the changes in stability and activity. Substitutions of catalytic residues Ser64, Lys67, Tyr150, Asn152, and Lys315 decrease the activity of the enzyme by 10(3)-10(5)-fold compared to wild-type. Concomitantly, many of these substitutions increase the stability of the enzyme significantly, by up to 4.7kcal/mol. To determine the structural origins of stabilization, the crystal structures of four mutant enzymes were determined to between 1.90A and 1.50A resolution. These structures revealed several mechanisms by which stability was increased, including mimicry of the substrate by the substituted residue (S64D), relief of steric strain (S64G), relief of electrostatic strain (K67Q), and improved polar complementarity (N152H). These results suggest that the preorganization of functionality characteristic of active sites has come at a considerable cost to enzyme stability. In proteins of unknown function, the presence of such destabilized regions may indicate the presence of a binding site.     

Control of coenzyme binding to horse liver alcohol dehydrogenase.

The rate of association of NAD(+) with wild-type horse liver alcohol dehydrogenase (ADH) is maximal at pH values between pK values of about 7 and 9, and the rate of NADH association is maximal at a pH below a pK of 9. The catalytic zinc-bound water, His-51 (which interacts with the 2'- and 3'-hydroxyl groups of the nicotinamide ribose of the coenzyme in the proton relay system), and Lys-228 (which interacts with the adenosine 3'-hydroxyl group and the pyrophosphate of the coenzyme) may be responsible for the observed pK values. In this study, the Lys228Arg, His51Gln, and Lys228Arg/His51Gln (to isolate the effect of the catalytic zinc-bound water) mutations were used to test the roles of the residues in coenzyme binding. The steady state kinetic constants at pH 8 for the His51Gln enzyme are similar to those for wild-type ADH. The Lys228Arg and Lys228Arg/His51Gln substitutions decrease the affinity for the coenzymes up to 16-fold, probably due to altered interactions with the arginine at position 228. As determined by transient kinetics, the rate constant for association of NAD(+) with the mutated enzymes no longer decreases at high pH. The pH profile for the Lys228Arg enzyme retains the pK value near 7. The His51Gln and Lys228Arg/His51Gln substitutions significantly decrease the rate constants for NAD(+) association, and the pH dependencies show that these enzymes bind NAD(+) most rapidly at a pH above pK values of 8. 0 and 9.0, respectively. It appears that the pK of 7 in the wild-type enzyme is shifted up by the H51Q substitutions, and the resulting pH dependence is due to the deprotonation of the catalytic zinc-bound water. Kinetic simulations suggest that isomerization of the enzyme-NAD(+) complex is substantially altered by the mutations. In contrast, the pH dependencies for NADH association with His51Gln, Lys228Arg, and Lys228Arg/His51Gln enzymes were the same as for wild-type ADH, suggesting that the binding of NAD(+) and the binding of NADH are controlled differently.     

Dissecting the electrostatic interactions and pH-dependent activity of a family 11 glycosidase

Previous studies of the low molecular mass family 11 xylanase from Bacillus circulans show that the ionization state of the nucleophile (Glu78, pK(a) 4.6) and the acid/base catalyst (Glu172, pK(a) 6.7) gives rise to its pH-dependent activity profile. Inspection of the crystal structure of BCX reveals that Glu78 and Glu172 are in very similar environments and are surrounded by several chemically equivalent and highly conserved active site residues. Hence, there are no obvious reasons why their apparent pK(a) values are different. To address this question, a mutagenic approach was implemented to determine what features establish the pK(a) values (measured directly by (13)C NMR and indirectly by pH-dependent activity profiles) of these two catalytic carboxylic acids. Analysis of several BCX variants indicates that the ionized form of Glu78 is preferentially stabilized over that of Glu172 in part by stronger hydrogen bonds contributed by two well-ordered residues, namely, Tyr69 and Gln127. In addition, theoretical pK(a) calculations show that Glu78 has a lower pK(a) value than Glu172 due to a smaller desolvation energy and more favorable background interactions with permanent partial charges and ionizable groups within the protein. The pK(a) value of Glu172 is in turn elevated due to electrostatic repulsion from the negatively charged glutamate at position 78. The results also indicate that all of the conserved active site residues act concertedly in establishing the pK(a) values of Glu78 and Glu172, with no particular residue being singly more important than any of the others. In general, residues that contribute positive charges and hydrogen bonds serve to lower the pK(a) values of Glu78 and Glu172. The degree to which a hydrogen bond lowers a pK(a) value is largely dependent on the length of the hydrogen bond (shorter bonds lower pK(a) values more) and the chemical nature of the donor (COOH > OH > CONH(2)). In contrast, neighboring carboxyl groups can either lower or raise the pK(a) values of the catalytic glutamic acids depending upon the electrostatic linkage of the ionization constants of the residues involved in the interaction. While the pH optimum of BCX can be shifted from -1.1 to +0.6 pH units by mutating neighboring residues within the active site, activity is usually compromised due to the loss of important ground and/or transition state interactions. These results suggest that the pH optima of an enzyme might be best engineered by making strategic amino acid substitutions, at positions outside of the "core" active site, that electrostatically influence catalytic residues without perturbing their immediate structural environment.     

Site-directed mutagenesis study of the microenvironment characteristics of Lys213 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate and carbon dioxide, and uses Mn(2+) as the activating metal ion. Comparison with the crystalline structure of homologous Escherichia coli PEP carboxykinase [Tari et al. Nature Struct. Biol. 4 (1997) 990-994] shows that Lys(213) is one of the ligands to Mn(2+) at the enzyme active site. Coordination of Mn(2+) to a lysyl residue is infrequent and suggests a low pK(a) value for the epsilon-NH(2) group of Lys(213). In this work, we evaluate the role of neighboring Phe(416) in contributing to provide a low polarity microenvironment suitable to keep the epsilon-NH(2) of Lys(213) in the unprotonated form. Mutation Phe416Tyr shows that the introduction of a hydroxyl group in the lateral chain of the residue produces a substantial loss in the enzyme affinity for Mn(2+), suggesting an increase of the pK(a) of Lys(213). A study of the effect of pH on K(m) for Mn(2+) indicate that the affinity of recombinant wild type enzyme for the metal ion is dependent on deprotonation of a group with pK(a) of 7.1+/-0.2, compatible with the low pK(a) expected for Lys(213). This pK(a) value increases at least 1.5 pH units upon Phe416Tyr mutation, in agreement with the expected effect of an increase in the polarity of Lys(213) microenvironment. Theoretical calculations of the pK(a) of Lys(213) indicate a value of 6.5+/-0.9, and it increases to 8.2+/-1.6 upon Phe416Tyr mutation. Additionally, mutation Phe416Tyr causes a loss of 1.3 kcal mol(-1) in the affinity of the enzyme for PEP, an effect perhaps related to the close proximity of Phe(416) to Arg(70), a residue previously shown to be important for PEP binding.     

Identification of lysine-78 as an essential residue in the Saccharomyces cerevisiae xylose reductase

Yeast xylose reductases are hypothesized as hybrid enzymes as their primary sequences contain elements of both the aldo-keto reductases (AKR) and short chain dehydrogenase/reductase (SDR) enzyme families. During catalysis by members of both enzyme families, an essential Lys residue H-bonds to a Tyr residue that donates proton to the aldehyde substrate. In the Saccharomyces cerevisiae xylose reductase, Tyr49 has been identified as the proton donor. However, the primary sequence of the enzyme contains two Lys residues, Lys53 and Lys78, corresponding to the conserved motifs for SDR and AKR enzyme families, respectively, that may H-bond to Tyr49. We used site-directed mutagenesis to substitute each of these Lys residues with Met. The activity of the K53M variant was slightly decreased as compared to the wild-type, while that of the K78M variant was negligible. The results suggest that Lys78 is the essential residue that H-bonds to Tyr49 during catalysis and indicate that the active site residues of yeast xylose reductases match those of the AKR, rather than SDR, enzymes. Intrinsic enzyme fluorescence spectroscopic analysis suggests that Lys78 may also contribute to the efficient binding of NADPH to the enzyme.