Mutagenesis of Trp(54) and Trp(203) residues on Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase significantly affects catalytic activities of the enzyme

The possible structural and catalytic functions of the nine tryptophan amino acid residues, including Trp(54), Trp(105), Trp(112), Trp(141), Trp(148), Trp(165), Trp(186), Trp(198), and Trp(203) in Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (Fs beta-glucanase), were characterized using site-directed mutagenesis, initial rate kinetics, fluorescence spectrometry, and structural modeling analysis. Kinetic studies showed that a 5-7-fold increase in K(m) value for lichenan was observed for W141F, W141H, and W203R mutant Fs beta-glucanases, and approximately 72-, 56-, 30-, 29.5-, 4.9-, and 4.3-fold decreases in k(cat) relative to that for the wild-type enzyme were observed for the W54F, W54Y, W141H, W203R, W141F, and W148F mutants, respectively. In contrast, W186F and W203F, unlike the other 12 mutants, exhibited a 1.4- and 4.2-fold increase in k(cat), respectively. W165F and W203R were the only two mutants that exhibited a 4-7-fold higher activity relative to the wild-type enzyme after they were incubated at pH 3.0 for 1 h. Fluorescence spectrometry indicated that all of the mutations on the nine tryptophan amino acid residues retained a folding similar to that of the wild-type enzyme. Structural modeling and kinetic studies suggest that Trp(54), Trp(141), Trp(148), and Trp(203) play important roles in maintaining structural integrity in the substrate-binding cleft and the catalytic efficiency of the enzyme.     

The role of tryptophan-48 in catalysis and binding of inhibitors of Plasmodium falciparum dihydrofolate reductase

Dihydrofolate reductases (DHFRs) from Plasmodium falciparum (Pf) and various species of both prokaryotic and eukaryotic organisms have a conserved tryptophan (Trp) at position 48 in the active site. The role in catalysis and binding of inhibitors of the conserved Trp48 of PfDHFR has been analysed by site-specific mutagenesis, enzyme kinetics and use of a bacterial surrogate system. All 19 mutant enzymes showed undetectable or very low specific activities, with the highest value of k(cat)/K(m) from the Tyr48 (W48Y) mutant (0.12 versus 11.94M(-1)s(-1)), of about 1% of the wild-type enzyme. The inhibition constants for pyrimethamine, cycloguanil and WR99210 of the W48Y mutants are 2.5-5.3 times those of the wild-type enzyme. All mutants, except W48Y, failed to support the growth of Escherichia coli transformed with the parasite gene in the presence of trimethoprim, indicating the loss of functional activity of the parasite enzyme. Hence, Trp48 plays a crucial role in catalysis and inhibitor binding of PfDHFR. Interestingly, W48Y with an additional mutation at Asn188Tyr (N188Y) was found to promote bacterial growth and yielded a higher amount of purified enzyme. However, the kinetic parameters of the purified W48Y+N188Y enzyme were comparable with W48Y and the binding affinities for DHFR inhibitors were also similar to the wild-type enzyme. Due to its conserved nature, Trp48 of PfDHFR is a potential site for interaction with antimalarial inhibitors which would not be compromised by its mutations.     

Adaptation of a thermophilic enzyme, 3-isopropylmalate dehydrogenase, to low temperatures

Random mutagenesis coupled with screening of the active enzyme at a low temperature was applied to isolate cold-adapted mutants of a thermophilic enzyme. Four mutant enzymes with enhanced specific activities (up to 4.1-fold at 40 degrees C) at a moderate temperature were isolated from randomly mutated Thermus thermophilus 3-isopropylmalate dehydrogenase. Kinetic analysis revealed two types of cold-adapted mutants, i.e. k(cat)-improved and K(m)-improved types. The k(cat)-improved mutants showed less temperature-dependent catalytic properties, resulting in improvement of k(cat) (up to 7.5-fold at 40 degrees C) at lower temperatures with increased K(m) values mainly for NAD. The K(m)-improved enzyme showed higher affinities toward the substrate and the coenzyme without significant change in k(cat) at the temperatures investigated (30-70 degrees C). In k(cat)-improved mutants, replacement of a residue was found near the binding pocket for the adenine portion of NAD. Two of the mutants retained thermal stability indistinguishable from the wild-type enzyme. Extreme thermal stability of the thermophilic enzyme is not necessarily decreased to improve the catalytic function at lower temperatures. The present strategy provides a powerful tool for obtaining active mutant enzymes at lower temperatures. The results also indicate that it is possible to obtain cold-adapted mutant enzymes with high thermal stability.     

Expression and characterization of active site mutants of hevamine, a chitinase from the rubber tree Hevea brasiliensis.

Hevamine is a chitinase from the rubber tree Hevea brasiliensis. Its active site contains Asp125, Glu127, and Tyr183, which interact with the -1 sugar residue of the substrate. To investigate their role in catalysis, we have successfully expressed wild-type enzyme and mutants of these residues as inclusion bodies in Escherichia coli. After refolding and purification they were characterized by both structural and enzyme kinetic studies. Mutation of Tyr183 to phenylalanine produced an enzyme with a lower k(cat) and a slightly higher K(m) than the wild-type enzyme. Mutating Asp125 and Glu127 to alanine gave mutants with approximately 2% residual activity. In contrast, the Asp125Asn mutant retained substantial activity, with an approximately twofold lower k(cat) and an approximately twofold higher K(m) than the wild-type enzyme. More interestingly, it showed activity to higher pH values than the other variants. The X-ray structure of the Asp125Ala/Glu127Ala double mutant soaked with chitotetraose shows that, compared with wild-type hevamine, the carbonyl oxygen atom of the N-acetyl group of the -1 sugar residue has rotated away from the C1 atom of that residue. The combined structural and kinetic data show that Asp125 and Tyr183 contribute to catalysis by positioning the carbonyl oxygen of the N-acetyl group near to the C1 atom. This allows the stabilization of a positively charged transient intermediate, in agreement with a previous proposal that the enzyme makes use of substrate-assisted catalysis.     

Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis. Gene cloning and protein characterization.

Random PCR mutagenesis was applied to the Thermus thermophilus xylA gene encoding xylose isomerase. Three cold-adapted mutants were isolated with the following amino-acid substitutions: E372G, V379A (M-1021), E372G, F163L (M-1024) and E372G (M-1026). The wild-type and mutated xylA genes were cloned and expressed in Escherichia coli HB101 using the vector pGEM-T Easy, and their physicochemical and catalytic properties were determined. The optimum pH for xylose isomerization activity for the mutants was approximately 7.0, which is similar to the wild-type enzyme. Compared with the wild-type, the mutants were active over a broader pH range. The mutants exhibited up to nine times higher catalytic rate constants (k(cat)) for d-xylose compared with the wild-type enzyme at 60 degrees C, but they did not show any increase in catalytic efficiency (k(cat)/K(m)). For d-glucose, both the k(cat) and the k(cat)/K(m) values for the mutants were increased compared with the wild-type enzyme. Furthermore, the mutant enzymes exhibited up to 255 times higher inhibition constants (K(i)) for xylitol than the wild-type, indicating that they are less inhibited by xylitol. The thermal stability of the mutated enzymes was poorer than that of the wild-type enzyme. The results are discussed in terms of increased molecular flexibility of the mutant enzymes at low temperatures.     

Entropy effects on protein hinges: the reaction catalyzed by triosephosphate isomerase

Many proteins utilize segmental motions to catalyze a specific reaction. The Omega loop of triosephosphate isomerase (TIM) is important for preventing the loss of the reactive enediol(ate) intermediate. The loop opens and closes even in the absence of the ligand, and the loop itself does not change conformation during movement. The conformational changes are localized to two hinges at the loop termini. Glycine is never observed in native TIM hinge sequences. In this paper, the hypothesis that limited access to conformational space is a requirement for protein hinges involved in catalysis was tested. The N-terminal hinge was mutated to P166/V167G/W168G (PGG), and the C-terminal hinge was mutated to K174G/T175G/A176G (GGG) in chicken TIM. The single-hinge mutants PGG and GGG had k(cat) values 200-fold lower than that of the wild type and K(m) values 10-fold higher. The k(cat) of double-hinge mutant P166/V167G/W168G/K174G/T175G/A176G was reduced 2500-fold; the K(m) was 10-fold higher. A combination of primary kinetic isotope effect measurements, isothermal calorimetric measurements, and (31)P NMR spectroscopic titration with the inhibitor 2-phosphoglycolate revealed that the mutants have a different ligand-binding mode than that of the wild-type enzyme. The predominant conformations of the mutants even in the presence of the inhibitor are loop-open conformations. In conclusion, mutation of the hinge residues to glycine resulted in the sampling of many more hinge conformations with the consequence that the population of the active-closed conformation is reduced. This reduced population results in a reduced catalytic activity.     

Arginine 49 is a bifunctional residue important in catalysis and biosynthesis of monomeric sarcosine oxidase: a context-sensitive model for the electrostatic impact of arginine to lysine mutations

Monomeric sarcosine oxidase (MSOX) contains covalently bound FAD and catalyzes the oxidative demethylation of sarcosine ( N-methylglycine). The side chain of Arg49 is in van der Waals contact with the si face of the flavin ring; sarcosine binds just above the re face. Covalent flavin attachment requires a basic residue (Arg or Lys) at position 49. Although flavinylation is scarcely affected, mutation of Arg49 to Lys causes a 40-fold decrease in k cat and a 150-fold decrease in k cat/ K m sarcosine. The overall structure of the Arg49Lys mutant is very similar to wild-type MSOX; the side chain of Lys49 in the mutant is nearly congruent to that of Arg49 in the wild-type enzyme. The Arg49Lys mutant exhibits several features consistent with a less electropositive active site: (1) Charge transfer bands observed for mutant enzyme complexes with competitive inhibitors absorb at higher energy than the corresponding wild-type complexes. (2) The p K a for ionization at N(3)H of FAD is more than two pH units higher in the mutant than in wild-type MSOX. (3) The reduction potential of the oxidized/radical couple in the mutant is 100 mV lower than in the wild-type enzyme. The lower reduction potential is likely to be a major cause of the reduced catalytic activity of the mutant. Electrostatic interactions with Arg49 play an important role in catalysis and covalent flavinylation. A context-sensitive model for the electrostatic impact of an arginine to lysine mutation can account for the dramatically different consequences of the Arg49Lys mutation on MSOX catalysis and holoenzyme biosysnthesis.     

Role of glutamate 64 in the activation of the prodrug 5-fluorocytosine by yeast cytosine deaminase

Yeast cytosine deaminase (yCD) catalyzes the hydrolytic deamination of cytosine to uracil as well as the deamination of the prodrug 5-fluorocytosine (5FC) to the anticancer drug 5-fluorouracil. In this study, the role of Glu64 in the activation of the prodrug 5FC was investigated by site-directed mutagenesis, biochemical, nuclear magnetic resonance (NMR), and computational studies. Steady-state kinetics studies showed that the mutation of Glu64 causes a dramatic decrease in k(cat) and a dramatic increase in K(m), indicating Glu64 is important for both binding and catalysis in the activation of 5FC. (19)F NMR experiments showed that binding of the inhibitor 5-fluoro-1H-pyrimidin-2-one (5FPy) to the wild-type yCD causes an upfield shift, indicating that the bound inhibitor is in the hydrated form, mimicking the transition state or the tetrahedral intermediate in the activation of 5FC. However, binding of 5FPy to the E64A mutant enzyme causes a downfield shift, indicating that the bound 5FPy remains in an unhydrated form in the complex with the mutant enzyme. (1)H and (15)N NMR analysis revealed trans-hydrogen bond D/H isotope effects on the hydrogen of the amide of Glu64, indicating that the carboxylate of Glu64 forms two hydrogen bonds with the hydrated 5FPy. ONIOM calculations showed that the wild-type yCD complex with the hydrated form of the inhibitor 1H-pyrimidin-2-one is more stable than the initial binding complex, and in contrast, with the E64A mutant enzyme, the hydrated inhibitor is no longer favored and the conversion has a higher activation energy, as well. The hydrated inhibitor is stabilized in the wild-type yCD by two hydrogen bonds between it and the carboxylate of Glu64 as revealed by (1)H and (15)N NMR analysis. To explore the functional role of Glu64 in catalysis, we investigated the deamination of cytosine catalyzed by the E64A mutant by ONIOM calculations. The results showed that without the assistance of Glu64, both proton transfers before and after the formation of the tetrahedral reaction intermediate become partially rate-limiting steps. The results of the experimental and computational studies together indicate that Glu64 plays a critical role in both the binding and the chemical transformation in the conversion of the prodrug 5FC to the anticancer drug 5-fluorouracil.     

Kinetic, stereochemical, and structural effects of mutations of the active site arginine residues in 4-oxalocrotonate tautomerase.

Three arginine residues (Arg-11, Arg-39, Arg-61) are found at the active site of 4-oxalocrotonate tautomerase in the X-ray structure of the affinity-labeled enzyme [Taylor, A. B., Czerwinski, R. M., Johnson, R. M., Jr., Whitman, C. P., and Hackert, M. L. (1998) Biochemistry 37, 14692-14700]. The catalytic roles of these arginines were examined by mutagenesis, kinetic, and heteronuclear NMR studies. With a 1,6-dicarboxylate substrate (2-hydroxymuconate), the R61A mutation showed no kinetic effects, while the R11A mutation decreased k(cat) 88-fold and increased K(m) 8.6-fold, suggesting both binding and catalytic roles for Arg-11. With a 1-monocarboxylate substrate (2-hydroxy-2,4-pentadienoate), no kinetic effects of the R11A mutation were found, indicating that Arg-11 interacts with the 6-carboxylate of the substrate. The stereoselectivity of the R11A-catalyzed protonation at C-5 of the dicarboxylate substrate decreased, while the stereoselectivity of protonation at C-3 of the monocarboxylate substrate increased in comparison with wild-type 4-OT, indicating the importance of Arg-11 in properly orienting the dicarboxylate substrate by interacting with the charged 6-carboxylate group. With 2-hydroxymuconate, the R39A and R39Q mutations decreased k(cat) by 125- and 389-fold and increased K(m) by 1.5- and 2.6-fold, respectively, suggesting a largely catalytic role for Arg-39. The activity of the R11A/R39A double mutant was at least 10(4)-fold lower than that of the wild-type enzyme, indicating approximate additivity of the effects of the two arginine mutants on k(cat). For both R11A and R39Q, 2D (1)H-(15)N HSQC and 3D (1)H-(15)N NOESY-HSQC spectra showed chemical shift changes mainly near the mutated residues, indicating otherwise intact protein structures. The changes in the R39Q mutant were mainly in the beta-hairpin from residues 50 to 57 which covers the active site. HSQC titration of R11A with the substrate analogue cis, cis-muconate yielded a K(d) of 22 mM, 37-fold greater than the K(d) found with wild-type 4-OT (0.6 mM). With the R39Q mutant, cis, cis-muconate showed negative cooperativity in active site binding with two K(d) values, 3.5 and 29 mM. This observation together with the low K(m) of 2-hydroxymuconate (0.47 mM) suggests that only the tight binding sites function catalytically in the R39Q mutant. The (15)Nepsilon resonances of all six Arg residues of 4-OT were assigned, and the assignments of Arg-11, -39, and -61 were confirmed by mutagenesis. The binding of cis,cis-muconate to wild-type 4-OT upshifts Arg-11 Nepsilon (by 0.05 ppm) and downshifts Arg-39 Nepsilon (by 1.19 ppm), indicating differing electronic delocalizations in the guanidinium groups. A mechanism is proposed in which Arg-11 interacts with the 6-carboxylate of the substrate to facilitate both substrate binding and catalysis and Arg-39 interacts with the 1-carboxylate and the 2-keto group of the substrate to promote carbonyl polarization and catalysis, while Pro-1 transfers protons from C-3 to C-5. This mechanism, together with the effects of mutations of catalytic residues on k(cat), provides a quantitative explanation of the 10(7)-fold catalytic power of 4-OT. Despite its presence in the active site in the crystal structure of the affinity-labeled enzyme, Arg-61 does not play a significant role in either substrate binding or catalysis.     

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.     

The contribution of tryptophan residues to conformational changes in clostridial glutamate dehydrogenase--W64 and W449 as mediators of the cooperative response to glutamate

The hexameric glutamate dehydrogenase of Clostridium symbiosum has previously been shown to undergo a pH-dependent inactivating conformational change that perturbs the environment of one or more Trp residues and is reversed by glutamate in a highly cooperative fashion with a Hill coefficient of almost 6. Five single mutants have now been made in which each of the Trp residues in turn has been replaced by Phe. All five were successfully over-produced as soluble proteins and purified. Far-UV CD showed that none of the mutations significantly affected secondary structure. All five proteins were active, ranging from 13 U.mg(-1) (W64F) to 20.8 U.mg(-1) (W393F), compared to 20 U.mg(-1) for wild-type, and the kinetic parameters at pH 7 were little changed, except for a five- to six-fold increase in Km for glutamate in W243F. Thermostability was also relatively little changed, although W310F and W393F were somewhat more stable and W64F less stable than the unmutated enzyme. All still showed the characteristic reversible, time-dependent high-pH inactivation. Near-UV CD spectra, reflecting the environment of aromatic residues, were recorded at both pH 7 and 8.8, and four of the mutants showed essentially the same perturbation in the 280 nm region as the wild-type enzyme. W64F, however, showed essentially no change. W64 is thus clearly a passive reporter of the pH-dependent conformational change, and not actually required for the transition to occur. The CD comparisons also suggest that the aromatic CD spectrum is contributed almost entirely by W64 and W449. Consistent with the pH-dependent change, all five mutant proteins also showed a positively cooperative response to glutamate at pH 9, reversing the inactivation. However, the Hill coefficient decreased from > 5 for wild-type to approximately 3 for the active site cleft mutation W243F and to approximately 2 for the interfacial mutants W64F and W449F in which the trimer-trimer interaction may be directly interrupted. W64 of each subunit is in contact with W449 in its dimer partner at the trimer-trimer interface. It seems that, although neither of these two residues is required for the pH-dependent change, together, they are essential in mediating the total cooperativity of the hexameric enzyme's response to glutamate and are presumably directly involved in transmitting conformational information between the two trimers.     

Engineering the substrate specificity of Alcaligenes D-aminoacylase useful for the production of D-amino acids by optical resolution

D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (AxD-NAase) offers a novel biotechnological application, the production of D-amino acid from the racemic mixture of N-acyl-DL-amino acids. However, its substrate specificity is biased toward certain N-acyl-D-amino acids. To construct mutant AxD-NAases with substrate specificities different from those of wild-type enzyme, the substrate recognition site of the AxD-NAase was rationally manipulated based on computational structural analysis and comparison of its primary structure with other D-aminoacylases with distinct substrate specificities. Mutations of amino acid residues, Phe191, Leu298, Tyr344, and Met346, which interact with the side chain of the substrate, induced marked changes in activities toward each substrate. For example, the catalytic efficiency (k(cat)/K(m)) of mutant F191W toward N-acetyl-D-Trp and N-acetyl-D-Ala was enhanced by 15.6- and 1.5-folds, respectively, compared with that of the wild-type enzyme, and the catalytic efficiency (k(cat)/K(m)) of mutant L298A toward N-acetyl-D-Trp was enhanced by 4.4-folds compared with that of the wild-type enzyme. Other enzymatic properties of both mutants, such as pH and temperature dependence, were the same as those of the wild-type enzyme. The F191W mutant in particular is considered to be useful for the enzymatic production of D-Trp which is an important building block of some therapeutic drugs.     

Aromatic stacking as a determinant of the thermal stability of CYP119 from Sulfolobus solfataricus

Two notable features of the thermophilic CYP119, an Arg154-Glu212 salt bridge between the F-G loop and the I helix and an extended aromatic cluster, were studied to determine their contributions to the thermal stability of the enzyme. Site-specific mutants of the salt bridge (Arg154, Glu212) and aromatic cluster (Tyr2, Trp4, Trp231, Tyr250, Trp281) were expressed and purified. The substrate-binding and kinetic constants for lauric acid hydroxylation are little affected in most mutants, but the E212D mutant is inactive and the R154Q mutant has higher K(s),K(m), and k(cat) values. The salt bridge mutants, like wild-type CYP119, melt at 91+/-1 degrees C, whereas mutation of individual residues in the extended aromatic cluster lowers the T(m) by 10-15 degrees C even though no change is observed on mutation of an unrelated aromatic residue. The extended aromatic cluster, but not the Arg154-Glu212 salt bridge, contributes to the thermal stability of CYP119.     

Contribution of W229 to the transglycosylation activity of 4-alpha-glucanotransferase from Pyrococcus furiosus

A W229H mutant of 4-alpha-glucanotransferase (4-alpha-GTase) from Pyrococcus furiosus was constructed and its catalytic properties were studied to investigate the role of W229 in the catalytic specificities of the enzyme. Various activities and kinetic parameters were determined for the wild-type and W229H mutant enzymes. The transglycosylation factor and transglycosylation activity of the mutant enzyme markedly decreased, but its hydrolysis activity was scarcely affected. It was discovered that the k(cat)/K(m) value of transglycosylation activity significantly decreased to about 15% of that of the wild type, while k(cat)/K(m) value of hydrolysis activity changed little for the mutant enzyme. The hydrophobicity of W229 was thought to be critical to the transglycosylation activity of the enzyme based on the enzyme's modeled tertiary structures.     

Biochemical characterization of recombinant acetyl xylan esterase from Aspergillus awamori expressed in Pichia pastoris: mutational analysis of catalytic residues

We engineered an acetyl xylan esterase (AwaxeA) gene from Aspergillus awamori into a heterologous expression system in Pichia pastoris. Purified recombinant AwAXEA (rAwAXEA) displayed the greatest hydrolytic activity toward alpha-naphthylacetate (C2), lower activity toward alpha-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. Putative catalytic residues, Ser(119), Ser(146), Asp(168) and Asp(202), were substituted for alanine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the four mutant enzymes were examined. The S119A and D202A mutant enzymes were catalytically inactive, whereas S146A and D168A mutants displayed significant hydrolytic activity. These observations indicate that Ser(119) and Asp(202) are important for catalysis. The S146A mutant enzyme showed lower specific activity toward the C2 substrate and higher thermal stability than wild-type enzyme. The lower activity of S146A was due to a combination of increased K(m) and decreased k(cat). The catalytic efficiency of S146A was 41% lower than that of wild-type enzyme. The synthesis of ethyl acetate was >10-fold than that of ethyl n-hexanoate synthesis for the wild-type, S146A and D168A mutant enzymes. However, the D202A showed greater synthetic activity of ethyl n-hexanoate as compared with the wild-type and other mutants.     

Tyrosine residues serve as proton donor in the catalytic mechanism of epoxide hydrolase from Agrobacterium radiobacter

Epoxide hydrolase from Agrobacterium radiobacter catalyzes the hydrolysis of epoxides to their diols via an alkyl-enzyme intermediate. The recently solved X-ray structure of the enzyme shows that two tyrosine residues (Tyr152 and Tyr215) are positioned close to the nucleophile Asp107 in such a way that they can serve as proton donor in the alkylation reaction step. The role of these tyrosines, which are conserved in other epoxide hydrolases, was studied by site-directed mutagenesis. Mutation of Tyr215 to Phe and Ala and mutation of Tyr152 to Phe resulted in mutant enzymes of which the k(cat) values were only 2-4-fold lower than for wild-type enzyme, whereas the K(m) values for the (R)-enantiomers of styrene oxide and p-nitrostyrene oxide were 3 orders of magnitude higher than the K(m) values of wild-type enzyme, showing that the alkylation half-reaction is severely affected by the mutations. Pre-steady-state analysis of the conversion of (R)-styrene oxide by the Y215F and Y215A mutants showed that the 1000-fold elevated K(m) values were mainly caused by a 15-40-fold increase in K(S) and a 20-fold reduction in the rate of alkylation. The rates of hydrolysis of the alkyl-enzyme intermediates were not significantly affected by the mutations. The double mutant Y152F+Y215F showed only a low residual activity for (R)-styrene oxide, with a k(cat)/K(m) value that was 6 orders of magnitude lower than with wild-type enzyme and 3 orders of magnitude lower than with the single tyrosine mutants. This indicates that the effects of the mutations were cumulative. The side chain of Gln134 is positioned in the active site of the X-ray structure of epoxide hydrolase. Mutation of Gln134 to Ala resulted in an active enzyme with slightly altered steady-state kinetic parameters compared to wild-type enzyme, indicating that Gln134 is not essential for catalysis and that the side chain of Gln134 mimics bound substrate. Based upon this observation, the inhibitory potential of various unsubstituted amides was tested, resulting in the identification of phenylacetamide as a competitive inhibitor with an inhibition constant of 30 microM.     

Trp(359) regulates flavin thermodynamics and coenzyme selectivity in Mycobacterium tuberculosis FprA

Mtb (Mycobacterium tuberculosis) FprA (flavoprotein reductase A) is an NAD(P)H-dependent FAD-binding reductase that is structurally related to mammalian adrenodoxin reductase, and which supports the catalytic function of Mtb cytochrome P450s. Trp(359), proximal to the FAD, was investigated in light of its potential role in controlling coenzyme interactions, as observed for similarly located aromatic residues in diflavin reductases. Phylogenetic analysis indicated that a tryptophan residue corresponding to Trp(359) is conserved across FprA-type enzymes and in adrenodoxin reductases. W359A/H mutants of Mtb FprA were generated, expressed and the proteins characterized to define the role of Trp(359). W359A/H mutants exhibited perturbed UV-visible absorption/fluorescence properties. The FAD semiquinone formed in wild-type NADPH-reduced FprA was destabilized in the W359A/H mutants, which also had more positive FAD midpoint reduction potentials (-168/-181 mV respectively, versus the standard hydrogen electrode, compared with -230 mV for wild-type FprA). The W359A/H mutants had lower ferricyanide reductase k(cat) and NAD(P)H K(m) values, but this led to improvements in catalytic efficiency (k(cat)/K(m)) with NADH as reducing coenzyme (9.6/18.8 muM(-1).min(-1) respectively, compared with 5.7 muM(-1).min(-1) for wild-type FprA). Stopped-flow spectroscopy revealed NAD(P)H-dependent FAD reduction as rate-limiting in steady-state catalysis, and to be retarded in mutants (e.g. limiting rate constants for NADH-dependent FAD reduction were 25.4 s(-1) for wild-type FprA and 4.8 s(-1)/13.4 s(-1) for W359A/H mutants). Diminished mutant FAD content (particularly in W359H FprA) highlighted the importance of Trp(359) for flavin stability. The results demonstrate that the conserved Trp(359) is critical in regulating FprA FAD binding, thermodynamic properties, catalytic efficiency and coenzyme selectivity.     

Role of Arg100 in the active site of adenosylcobalamin-dependent glutamate mutase

Arginine-100 is involved in recognizing the gamma carboxylate of the substrate in glutamate mutase. To investigate its role in substrate binding and catalysis, this residue was mutated to lysine, tyrosine, and methionine. The effect of these mutations was to reduce k(cat) by 120-320-fold and to increase K(m(apparent)) for glutamate by 13-22-fold; K(m(apparent)) for adenosylcobalamin is little changed by these mutations. Even at saturating substrate concentrations, no cob(II)alamin could be detected in the UV-visible spectra of the Arg100Tyr and Arg100Met mutants. However, in the Arg100Lys mutant cob(II)alamin accumulated to concentrations similar to wild-type enzyme, which allowed the pre-steady-state kinetics of adenosylcobalamin homolysis to be investigated by stopped-flow spectroscopy. It was found that homolysis of the coenzyme is slower by an order of magnitude, compared with wild-type enzyme. Furthermore, glutamate binding is significantly weakened, so much so that the reaction exhibits second-order kinetics over the range of substrate concentrations used. The Arg100Lys mutant does not exhibit the very large deuterium isotope effects that are observed for homolysis of the coenzyme when the wild-type enzyme is reacted with deuterated substrates; this suggests that homolysis is slowed relative to hydrogen abstraction by this mutation.     

Cofactor requirements of BamHI mutant endonuclease E77K and its suppressor mutants

A mutant BamHI endonuclease, E77K, belongs to a class of catalytic mutants that bind DNA efficiently but cleave DNA at a rate more than 10(3)-fold lower than that of the wild-type enzyme (S. Y. Xu and I. Schildkraut, J. Biol. Chem. 266:4425-4429, 1991). The preferred cofactor for the wild-type BamHI is Mg2+. BamHI is 10-fold less active with Mn2+ as the cofactor. In contrast, the E77K variant displays an increased activity when Mn2+ is substituted for Mg2+ in the reaction buffer. Mutations that partially suppress the E77K mutation were isolated by using an Escherichia coli indicator strain containing the dinD::lacZ fusion. These pseudorevertant endonucleases induce E. coli SOS response (as evidenced by blue colony formation) and thus presumably nick or cleave chromosomal DNA in vivo. Consistent with the in vivo result, the pseudorevertant endonucleases in the crude cell extract display site-specific partial DNA cleavage activity. DNA sequencing revealed two unique suppressing mutations that were located within two amino acid residues of the original mutation. Both pseudorevertant proteins were purified and shown to increase specific activity at least 50-fold. Like the wild-type enzyme, both pseudorevertant endonucleases prefer Mg2+ as the cofactor. Thus, the second-site mutation not only restores partial cleavage activity but also suppresses the metal preference as well. These results suggest that the Glu-77 residue may play a role in metal ion binding or in enzyme activation (allosteric transition) following sequence-specific recognition.     

Kinetic studies on drug-resistant variants of Escherichia coli thymidylate synthase: functional effects of amino acid substitutions at residue 4.

A naturally occurring mutant of human thymidylate synthase (hTS) that contains a Tyr to His mutation at residue 33 was found to confer 4-fold resistance to 5-fluoro-2'-deoxyuridine (FdUrd), a prodrug of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). The crystal structure of hTS implicated this Tyr residue in a drug resistance mechanistic role that may include both substrate binding and catalysis (Schiffer et al., Biochemistry, 34, 16279-16287, 1995). Because of the existence of a defined kinetic scheme and the development of a bacterial expression vector for the overproduction of Escherichia coli TS (ecTS), we chose to initially study the corresponding residue in the bacterial enzyme, Tyr 4 of ecTS. Nine mutant ecTS enzymes that differed in sequence at position 4 were generated. Mutants with a charged or polar side chain (Ser, Cys, Asp, and Arg) and Gly precipitated in the cell paste, resulting in no catalytic activity in cell-free extracts. Although most of the His 4 mutant precipitated, sufficient amounts remained in the cell-free extract to permit isolation to near homogeneity. Wild-type ecTS and mutants with a hydrophobic side chain (Phe, Ile, and Val) were expressed at nearly 30% of the total cellular protein. The k(cat) values for the isolatable mutants were 2- to 10-fold lower than that of the wild-type enzyme, while the K(m) values for 2'-deoxyuridylate (dUMP) and 5,10-methylenetetrahydrofolate (CH(2)H(4)folate) were similar for all the mutants. Dissociation constants for binary complex formation determined by stopped-flow spectroscopy were similar for the wild-type and mutant enzymes for both dUMP and 2'-deoxythymidylate, indicating that this mutation does not significantly alter the binding of the natural nucleotide ligands. However, each mutant enzyme had three- to 5-fold lower affinity for FdUMP in the binary complex compared with the wild-type enzyme, and only His 4 showed a lower affinity for FdUMP in the ternary complex. Analysis of k(burst) showed that the initial binding of CH(2)H(4)folate is weaker for each mutant compared to the wild-type enzyme and that lower k(cat) values were due to compromised rates that govern the chemical transformation of bound substrates to bound products.