Roles for the two cysteine residues of AhpC in catalysis of peroxide reduction by alkyl hydroperoxide reductase from Salmonella typhimurium

The catalytic properties of cysteine residues Cys46 and Cys165, which form intersubunit disulfide bonds in the peroxidatic AhpC protein of the alkyl hydroperoxide reductase (AhpR) system from Salmonella typhimurium, have been investigated. The AhpR system, composed of AhpC and a flavoprotein reductase, AhpF, catalyzes the pyridine nucleotide-dependent reduction of organic hydroperoxides and hydrogen peroxide. Amino acid sequence analysis of the disulfide-containing tryptic peptide demonstrated the presence of two identical disulfide bonds per dimer of oxidized AhpC located between Cys46 on one subunit and Cys165 on the other. Mutant AhpC proteins containing only one (C46S and C165S) or no (C46,165S) cysteine residues were purified and shown by circular dichroism studies to exhibit no major disruptions in secondary structure. In NADH-dependent peroxidase assays in the presence of AhpF, the C165S mutant was fully active in comparison with wild-type AhpC, while C46S and C46,165S displayed no peroxidatic activity. In addition, only C165S was oxidized by 1 equiv of hydrogen peroxide, giving a species that was stoichiometrically reducible by NADH in the presence of a catalytic amount of AhpF. Oxidized C165S also reacted rapidly with a stoichiometric amount of the thiol-containing reagent 2-nitro-5-thiobenzoic acid to generate a mixed disulfide, and was susceptible to inactivation by hydrogen peroxide, strongly supporting its identification as a cysteine sulfenic acid (Cys46-SOH). The lack of reactivity of the C46S mutant toward peroxides was not a result of inaccessibility of the remaining thiol as demonstrated by its modification with 5, 5'-dithiobis(2-nitrobenzoic acid), but could be due to the lack of a proximal active-site base which would support catalysis through proton donation to the poor RO- leaving group. Our results clearly identify Cys46 as the peroxidatic center of AhpC and Cys165 as an important residue for preserving the activity of wild-type AhpC by reacting with the nascent sulfenic acid of the oxidized protein (Cys46-SOH) to generate a stable disulfide bond, thus preventing further oxidation of Cys46-SOH by substrate.     

Mutational analysis of the major loop of Bacillus 1,3-1,4-beta-D-glucan 4-glucanohydrolases. Effects on protein stability and substrate binding

The carbohydrate-binding cleft of Bacillus licheniformis 1,3-1, 4-beta-D-glucan 4-glucanohydrolase is partially covered by the surface loop between residues 51 and 67, which is linked to beta-strand-(87-95) of the minor beta-sheet III of the protein core by a single disulfide bond at Cys61-Cys90. An alanine scanning mutagenesis approach has been applied to analyze the role of loop residues from Asp51 to Arg64 in substrate binding and stability by means of equilibrium urea denaturation, enzyme thermotolerance, and kinetics. The DeltaDeltaGU between oxidized and reduced forms is approximately constant for all mutants, with a contribution of 5.3 +/- 0.2 kcal.mol-1 for the disulfide bridge to protein stability. A good correlation is observed between DeltaGU values by reversible unfolding and enzyme thermotolerance. The N57A mutant, however, is more thermotolerant than the wild-type enzyme, whereas it is slightly less stable to reversible urea denaturation. Mutants with a <2-fold increase in Km correspond to mutations at residues not involved in substrate binding, for which the reduction in catalytic efficiency (kcat/Km) is proportional to the loss of stability relative to the wild-type enzyme. Y53A, N55A, F59A, and W63A, on the other hand, show a pronounced effect on catalytic efficiency, with Km > 2-fold and kcat < 5% of the wild-type values. These mutated residues are directly involved in substrate binding or in hydrophobic packing of the loop. Interestingly, the mutation M58A yields an enzyme that is more active than the wild-type enzyme (7-fold increase in kcat), but it is slightly less stable.     

Kinetic and spectroscopic investigations of wild-type and mutant forms of apple 1-aminocyclopropane-1-carboxylate synthase

Two catalytically inactive mutant forms of 1-aminocyclopropane-1-carboxylate (ACC) synthase, Y85A and K273A, were mixed in low concentrations of guanidine hydrochloride (GdnHCl). About 15% of the wild-type activity was recovered (theoretical 25% for a binomial distribution), proving that the functional unit of the enzyme is a dimer, or theoretically, a higher order oligomer. The enzyme catalyzes the conversion of S-adenosyl-L-methionine (SAM) to ACC. The value of kcat/KM is 1.2 x 10(6) M-1 s-1 at pH 8.3. Viscosity variation experiments with glycerol and sucrose as viscosogenic reagents showed that this reaction is nearly 100% diffusion controlled. The sensitivity to viscosity for the corresponding reaction of the less reactive Y233F mutant is much reduced, thus the latter reaction serves as a control for that of the wild-type enzyme. The kcat/KM vs pH profile for wild-type enzyme exhibits pKa values of 7.5 and 8.9. The former is assigned to the pKa of the alpha-amino group of SAM, while the latter corresponds to the independently determined spectrophotometric pKa of the internal aldimine. The kcat vs pH profile exhibits similar pKas, which means that the above pKa values are not perturbed in the Michaelis complex. The phenolic hydroxyl group of Tyr233 forms a hydrogen bond to the 3'-O- of PLP. The spectral and kinetic pKa (kcat/KM) values of the Y233F mutant are not identical (spectral 10.2, kinetic 8.7). A model that accounts quantitatively for these data posits two parallel pathways to the external aldimine for this mutant, the minor one has the alpha-amino group free base form of SAM reacting with the protonated imine form of the enzyme with kcat/KM approximately 6.0 x 10(3) M-1 s-1, while the major pathway involves reaction of the aldehyde form of PLP with SAM with kcat/KM approximately 7.0 x 10(5) M-1 s-1. The spectral pKa is defined only by the less reactive species.     

Steady-state kinetic mechanism, stereospecificity, substrate and inhibitor specificity of Enterobacter cloacae nitroreductase

Enterobacter cloacae nitroreductase (NR) is a flavoprotein which catalyzes the pyridine nucleotide-dependent reduction of nitroaromatics. Initial velocity and inhibition studies have been performed which establish unambiguously a ping-pong kinetic mechanism. NADH oxidation proceeds stereospecifically with the transfer of the pro-R hydrogen to the enzyme and the amide moiety of the nicotinamide appears to be the principal mediator of the interaction between NR and NADH. 2,4-Dinitrotoluene is the most efficient oxidizing substrate examined, with a kcat/KM an order of magnitude higher than those of p-nitrobenzoate, FMN, FAD or riboflavin. Dicoumarol is a potent inhibitor competitive vs. NADH with a Ki of 62 nM. Several compounds containing a carboxyl group are also competitive inhibitors vs. NADH. Yonetani-Theorell analysis of dicoumarol and acetate inhibition indicates that their binding is mutually exclusive, which suggests that the two inhibitors bind to the same site on the enzyme. NAD+ does not exhibit product inhibition and in the absence of an electron acceptor, no isotope exchange between NADH and 32P-NAD+ could be detected. NR catalyzes the 4-electron reduction of nitrobenzene to hydroxylaminobenzene with no optically detectable net formation of the putative two-electron intermediate nitrosobenzene.     

The Na(+)-translocating NADH:ubiquinone oxidoreductase from the marine bacterium Vibrio alginolyticus contains FAD but not FMN

The Na(+)-translocating NADH:ubiquinone oxidoreductase from Vibrio alginolyticus was extracted from the bacterial membranes and purified by ion exchange chromatographic procedures. The enzyme catalyzed NADH oxidation by suitable electron acceptors, e.g. menadione, and the Na+ and NADH-dependent reduction of ubiquinone-1. Four dominant bands and a number of minor bands were visible on SDS-PAGE that could be part of the enzyme complex. Flavin analyses indicated the presence of FAD but no FMN in the purified enzyme. FAD but no FMN were also present in V. alginolyticus membranes. FAD is therefore a prosthetic group of the Na(+)-translocating NADH:ubiquinone oxidoreductase and FMN is not present in the enzyme. The FAD was copurified with the NADH dehydrogenase. The purified enzyme exhibited an absorption spectrum with a maximum at 450 nm that is typical for a flavoprotein. Upon incubation with NADH this absorption disappeared indicating reduction of the enzyme-bound FAD.     

The role of cysteine residues of spinach ferredoxin-NADP+ reductase As assessed by site-directed mutagenesis

To investigate the functional role of the cysteine residues present in the spinach ferredoxin-NADP+ oxidoreductase, we individually replaced each of the five cysteine residues with serine using site-directed mutagenesis. All of the mutant reductases were correctly assembled in Escherichia coli except for the C42S mutant protein. C114S and C137S mutant enzymes apparently showed structural and kinetic properties very similar to those of the wild-type reductase. However, C272S and C132S mutations yielded enzymes with a decreased catalytic activity in the ferredoxin-dependent reaction (14 and 31% of the wild type, respectively). Whereas the C132S was fully competent in the diaphorase reaction, the C272S mutant flavoprotein showed a 35-fold reduction in catalytic efficiency with respect to the wild-type enzyme (0.4 versus 14.28 microM-1 s-1) due to a substantial decrease of kcat. NADP+ binding by the C272S mutant enzyme was apparently quantitatively the same (Kd = 37 microM) but qualitatively different, as shown by the differential spectrum. Stopped-flow experiments showed that the enzyme-FAD reduction rate was considerably decreased in the C272S mutant reductase, along with a much lower yield of the charge-transfer transient species. It is inferred from these data that the charge transfer (FAD-NADPH) between the reductase and NADPH is required for hydride transfer from the pyridine nucleotide to flavin to occur with a rate compatible with catalysis.     

Role of active site tyrosine residues in catalysis by human glutathione reductase

Tyr114 and Tyr197 are highly conserved residues in the active site of human glutathione reductase, Tyr114 in the glutathione disulfide (GSSG) binding site and Tyr197 in the NADPH site. Mutation of either residue has profound effects on catalysis. Y197S and Y114L have 17% and 14% the activity of the wild-type enzyme, respectively. Mutation of Tyr197, in the NADPH site, leads to a decrease in Km for GSSG, and mutation of Tyr114, in the GSSG site, leads to a decrease in Km for NADPH. This behavior is predicted for enzymes operating by a ping-pong mechanism where both half-reactions partially limit turnover. Titration of the wild-type enzyme or Y114L with NADPH proceeds in two phases, Eox to EH2 and EH2 to EH2-NADPH. In contrast, Y197S reacts monophasically, showing that excess NADPH fails to enhance the absorbance of the thiolate-FAD charge-transfer complex, the predominant EH2 form of glutathione reductase. The reductive half-reactions of the wild-type enzyme and of Y114L are similar; FAD reduction is fast (approximately 500 s-1 at 4 degreesC) and thiolate-FAD charge-transfer complex formation has a rate of 100 s-1. In Y197S, these rates are only 78 and 5 s-1, respectively. The oxidative half-reaction, the rate of reoxidation of EH2 by GSSG, of the wild-type enzyme is approximately 4-fold faster than that of Y114L. These results are consistent with Tyr197 serving as a gate in the binding of NADPH, and they indicate that Tyr114 assists the acid catalyst His467'.     

A flavoprotein functional as NADH oxidase from Amphibacillus xylanus Ep01: purification and characterization of the enzyme and structural analysis of its gene

Amphibacillus xylanus Ep01, a facultative anaerobe we recently isolated, shows rapid aerobic growth even though it lacks a respiratory pathway. Thus, the oxidative consumption of NADH, produced during glycolysis and pyruvate oxidation, should be especially important for maintenance of intracellular redox balance in this bacterium. We purified a flavoprotein functional as NADH oxidase from aerobically growing A. xylanus Ep01. The A. xylanus enzyme is a homotetramer composed of a subunit (M(r) 56,000) containing 1 mol of flavin adenine dinucleotide. This enzyme catalyzes the reduction of oxygen to hydrogen peroxide with beta-NADH as the preferred electron donor and exhibits no activity with NADPH. The flavoprotein gene of A. xylanus Ep01 was cloned by using a specific antibody. The amino acid sequence of 509 residues, deduced from the nucleotide sequence, showed 51.2 and 72.5% identities to the amino acid sequences of alkyl hydroperoxide reductase from Salmonella typhimurium and NADH dehydrogenase from alkalophilic Bacillus sp. strain YN-1, respectively. Bacillus spp. have a respiratory chain and grow well under aerobic conditions. In contrast, Amphibacillus spp., having no respiratory chain, grow equally well under both aerobic and anaerobic conditions, which distinguishes these two genera. Salmonella spp., which are gram-negative bacteria, are taxonomically distant from gram-positive bacteria such as Bacillus spp. and Amphibacillus spp. The above findings, however, suggest that the flavoprotein functional as NADH oxidase, the alkyl hydroperoxide reductase, and the NADH dehydrogenase diverged recently, with only small changes leading to their functional differences.     

Active site of dihydroorotate dehydrogenase A from Lactococcus lactis investigated by chemical modification and mutagenesis

The flavin-containing enzyme dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate (DHO) to orotate, the first aromatic intermediate in pyrimidine biosynthesis. The first structure of a DHOD, the A form of the enzyme from Lactococcus lactis, has recently become known, and some conserved residues were suggested to have a role in the active site [Rowland et al. (1997) Structure 2, 239-252]. In particular, Cys 130 was hypothesized to work as a base, which activates dihydroorotate (DHO) for hydride transfer. By chemical modification and site-directed mutagenesis we have obtained results consistent with this proposal. Cys 130 was susceptible to alkylating reagents, and mutants of Cys 130 (C130A and C130S) showed hardly detectable enzyme activity at pH 8.0, while at pH 10 the C130S mutant enzyme had approximately 1% of wild-type activity. Mutants of Lys 43, Asn 132, and Lys 164 were also constructed. Exchange of Lys 43 to Ala or Glu (K43A and K43E) and of Asn 132 to Ala (N132A) affected both catalysis and substrate binding. Expressed as kcat/KM for DHO, the deterioration of these three mutant enzymes was 10(3)-10(4)-fold. Flavin spectra of the mutant enzymes were not, like the wild-type enzyme, bleached by DHO in stopped-flow experiments, showing that they were deficient with respect to the first half-reaction, namely reduction of FMN by DHO, which was not rate limiting for the wild-type enzyme. The binding interaction between flavin and the reaction product, orotate, could be monitored by a red shift of the flavin absorbance in the wild-type enzyme. The C130A, C130S, and N132A mutant enzymes displayed similar capacity to bind orotate. In contrast, orotate did not change the absorption spectra of the K43 mutant enzymes, although it did inhibit their activity. All of the mutant enzymes, except K164A, contained normal levels of flavin. The results are discussed in relation to the structures of DHODA and other flavoenzymes. The possible acid-base chemistry of Cys 130 is compared to previous work on mammalian dihydropyrimidine dehydrogenases, flavoenzymes, which catalyze the reversed reaction, namely the reduction of pyrimidine bases.     

Porcine recombinant dihydropyrimidine dehydrogenase: comparison of the spectroscopic and catalytic properties of the wild-type and C671A mutant enzymes

Dihydropyrimidine dehydrogenase catalyzes, in the rate-limiting step of the pyrimidine degradation pathway, the NADPH-dependent reduction of uracil and thymine to dihydrouracil and dihydrothymine, respectively. The porcine enzyme is a homodimeric iron-sulfur flavoprotein (2 x 111 kDa). C671, the residue postulated to be in the uracil binding site and to act as the catalytically essential acidic residue of the enzyme oxidative half-reaction, was replaced by an alanyl residue. The mutant enzyme was overproduced in Escherichia coli DH5alpha cells, purified to homogeneity, and characterized in comparison with the wild-type species. An extinction coefficient of 74 mM-1 cm-1 was determined at 450 nm for the wild-type and mutant enzymes. Chemical analyses of the flavin, iron, and acid-labile sulfur content of the enzyme subunits revealed similar stoichiometries for wild-type and C671A dihydropyrimidine dehydrogenases. One FAD and one FMN per enzyme subunit were found. Approximately 16 iron atoms and 16 acid-labile sulfur atoms were found per wild-type and mutant enzyme subunit. The C671A dihydropyrimidine dehydrogenase mutant exhibited approximately 1% of the activity of the wild-type enzyme, thus preventing its steady-state kinetic analysis. Therefore, the ability of the C671A mutant and, for comparison, of the wild-type enzyme species to interact with reaction substrates, products, or their analogues were studied by absorption spectroscopy. Both enzyme forms did not react with sulfite. The wild-type and mutant enzymes were very similar to each other with respect to the spectral changes induced by binding of the reaction product NADP+ or of its nonreducible analogue 3-aminopyridine dinucleotide phosphate. Uracil also induced qualitatively and quantitatively similar absorbance changes in the visible region of the absorbance spectrum of the two enzyme forms. However, the calculated Kd of the enzyme-uracil complex was significantly higher for the C671A mutant (9.1 +/- 0.7 microM) than for the wild-type dihydropyrimidine dehydrogenase (0.7 +/- 0.09 microM). In line with these observations, the two enzyme forms behaved in a similar way when titrated anaerobically with a NADPH solution. Addition of an up to 10-fold excess of NADPH to both dihydropyrimidine dehydrogenase forms led to absorbance changes consistent with reduction of approximately 0.5 flavin per subunit, with no indication of reduction of the enzyme iron-sulfur clusters. Absorbance changes consistent with reduction of both enzyme flavins were obtained by removing NADP+ with a NADPH-regenerating system. On the contrary, the two enzyme species differed significantly with respect to their reactivity with dihydrouracil. Addition of dihydrouracil to the wild-type enzyme species, under anaerobic conditions, led to absorbance changes that could be interpreted to result from both partial flavin reduction and the formation of a complex between the enzyme and (dihydro)uracil. In contrast, only spectral changes consistent with formation of a complex between the oxidized enzyme and dihydrouracil were observed when a C671A mutant enzyme solution was titrated with this compound. Furthermore, enzyme-monitored turnover experiments were carried out anaerobically in the presence of a limiting amount of NADPH and excess uracil with the two enzyme forms in a stopped-flow apparatus. These experiments directly demonstrated that the substitution of an alanyl residue for C671 in dihydropyrimidine dehydrogenase specifically prevents enzyme-catalyzed reduction of uracil. Finally, sequence analysis of dihydropyrimidine dehydrogenase revealed that it exhibits a modular structure; the N-terminal region, similar to the beta subunit of bacterial glutamate synthases, is proposed to be responsible for NADPH binding and oxidation with reduction of the FAD cofactor of dihydropyrimidine dehydrogenase. The central region, similar to the FMN subunit of dihydroorotate dehydrogenases, is likely to harbor the site o     

Rapid reduction of Hg(II) by mercuric ion reductase does not require the conserved C-terminal cysteine pair using HgBr2 as the substrate

Conditions are described under which the nonphysiological substrate mercuric bromide (HgBr2) is rapidly turned over, both by the wild type (CCCC) and by an active site double mutant (CCAA) of mercuric reductase in which the C-terminal cysteines 557' and 558' are replaced by alanine and only the redox-active pair Cys135 and Cys140 are available for catalysis. A maximum rate of turnover kcatapp of approximately 18 s-1 (at 3 degreesC) for both enzymes is observed, and at high [HgBr2]/[enzyme] ratios, inhibition is found. The UV-vis spectral changes during turnover are closely similar in both enzymes, indicating that catalysis follows the same enzymatic mechanism. Single-turnover analysis of the mutant enzyme shows that after binding of HgBr2, two further rapid events ensue, followed by reduction of the metal ion (kobs approximately 23.5 s-1). It is shown that under multiple-turnover conditions, completion of the catalytic cycle must occur via an ordered mechanism where rapid binding of a new molecule of HgBr2 to EH2.NADP+ precedes exchange of the pyridine nucleotide. Binding of HgBr2 to the active site triple mutant C135A/C557A/C558A (ACAA) is ca. 100-fold slower compared to that of the CCAA mutant and results in no detectable turnover. It is concluded that in the reducible enzyme.Hg(II) complex, the metal ion is coordinated to Cys135 and Cys140 and that for efficient catalysis both residues are required. Furthermore, the data imply that binding to EH2.NADPH occurs via initial rate-limiting attack of Cys135, followed by reaction with Cys140.     

Hydrolysis of phosphodiesters through transformation of the bacterial phosphotriesterase

The phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of a wide array of phosphotriesters and related phosphonates, including organophosphate pesticides and military nerve agents. It has now been shown that this enzyme can also catalyze the hydrolysis of phosphodiesters, albeit at a greatly reduced rate. However, the enzymatic hydrolysis of ethyl-4-nitrophenyl phosphate (compound I) by the wild-type enzyme was >10(8) times faster than the uncatalyzed reaction (kcat = 0.06 s-1 and Km = 38 mM). Upon the addition of various alkylamines to the reaction mixture, the kcat/Km for the phosphodiester (compound I) increased up to 200-fold. Four mutant enzymes of the phosphotriesterase were constructed in a preliminary attempt to improve phosphodiester hydrolysis activity of the native enzyme. Met-317, which is thought to reside in close proximity to the pro-S-ethoxy arm of the paraoxon substrate, was mutated to arginine, alanine, histidine, and lysine. These mutant enzymes showed slight improvements in the catalytic hydrolysis of organophosphate diesters. The M317K mutant enzyme displayed the most improvement in catalytic activity (kcat = 0.34 s-1 and Km = 30 mM). The M317A mutant enzyme catalyzed the hydrolysis of the phosphodiester (compound I) in the presence of alkylamines up to 200 times faster than the wild-type enzyme in the absence of added amines. The neutralization of the negative charge on the oxygen atom of the phosphodiester by the ammonium cation within the active site is thought to be responsible for the rate enhancement by these amines in the hydrolytic reaction. These results demonstrate that an active site optimized for the hydrolysis of organophosphate triesters can be made to catalyze the hydrolysis of organophosphate diesters.     

Novel application of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole to identify cysteine sulfenic acid in the AhpC component of alkyl hydroperoxide reductase

The trapping of a sulfenic acid within the fully active C165S mutant of the AhpC peroxidase protein from Salmonella typhimurium was investigated. The electrophilic reagent employed in these studies, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl), has previously been used to modify thiol, amino, and tyrosine hydroxyl groups in proteins; at neutral pH only cysteinyl residues of AhpC proteins are modified. The peroxide-oxidized C165S mutant of AhpC incubated with NBD-Cl gave a product with an absorbance maximum at 347 nm, whereas the thiol-NBD conjugate formed from the reduced protein absorbed maximally at 420 nm. Electrospray ionization mass spectrometry of the modified proteins allowed identification of the species absorbing at 347 nm as a Cys-S(O)-NBD derivative containing one additional oxygen relative to the Cys-S-NBD product. The C165S conjugates with Cys-S(O)-NBD and Cys-S-NBD had no peroxidase activity when compared to unreacted C165S and wild-type AhpC, but were both reactivated through removal of NBD by DTT. Oxidized C165S was also modified by dimedone, a common sulfenic acid reagent, to give the expected inactivated conjugate of higher mass. This reagent was not removed by DTT and blocked any further reaction of the protein with NBD-Cl. NBD modification of Enterococcus faecalis NADH peroxidase, a well-characterized flavoprotein with an active-site sulfenic acid (Cys-SOH), also yielded the spectrally-distinguishable NBD conjugates following incubation of NBD-Cl with oxidized and reduced forms of the denatured peroxidase, indicating a general utility for this reagent with other sulfenic acid-containing proteins. A significant advantage of NBD-Cl over previously-used sulfenic acid reagents such as dimedone is in the retention of the sulfenic acid oxygen in the modified product; differentiation between protein-associated thiols and sulfenic acids is therefore now possible by means of both visible absorbance properties and mass analyses of the NBD-modified proteins.     

Effects of abnormal-sterol accumulation on Ustilago maydis plasma membrane H+-ATPase stoichiometry and polypeptide pattern

A flavoprotein with NADH oxidising activity (NADH: acceptor oxidoreductase) was isolated from the soluble fraction of the thermoacidophilic archaea Acidianus ambivalens. The protein is a monomer with a molecular mass of 70 kDa and contains FAD as single cofactor. Its activity as NADH:O2 oxidoreductase is FAD, but not FMN, dependent and yields hydrogen peroxide as the reaction product. The activity decreases with pH in the range 4.5 to 9.8, and increases with the temperature, as tested from 30 degrees to 60 degrees C. As elicited by EPR, the purified enzyme also acts as an NADH:ferredoxin oxidoreductase. These features are discussed in light of the possible involvement of this protein in the metabolism of this archaea.     

Active-site Arg --> Lys substitutions alter reaction and substrate specificity of aspartate aminotransferase

Arg386 and Arg292 of aspartate aminotransferase bind the alpha and the distal carboxylate group, respectively, of dicarboxylic substrates. Their substitution with lysine residues markedly decreased aminotransferase activity. The kcat values with L-aspartate and 2-oxoglutarate as substrates under steady-state conditions at 25 degrees C were 0.5, 2.0, and 0.03 s-1 for the R292K, R386K, and R292K/R386K mutations, respectively, kcat of the wild-type enzyme being 220 s-1. Longer dicarboxylic substrates did not compensate for the shorter side chain of the lysine residues. Consistent with the different roles of Arg292 and Arg386 in substrate binding, the effects of their substitution on the activity toward long chain monocarboxylic (norleucine/2-oxocaproic acid) and aromatic substrates diverged. Whereas the R292K mutation did not impair the aminotransferase activity toward these substrates, the effect of the R386K substitution was similar to that on the activity toward dicarboxylic substrates. All three mutant enzymes catalyzed as side reactions the beta-decarboxylation of L-aspartate and the racemization of amino acids at faster rates than the wild-type enzyme. The changes in reaction specificity were most pronounced in aspartate aminotransferase R292K, which decarboxylated L-aspartate to L-alanine 15 times faster (kcat = 0.002 s-1) than the wild-type enzyme. The rates of racemization of L-aspartate, L-glutamate, and L-alanine were 3, 5, and 2 times, respectively, faster than with the wild-type enzyme. Thus, Arg --> Lys substitutions in the active site of aspartate aminotransferase decrease aminotransferase activity but increase other pyridoxal 5'-phosphate-dependent catalytic activities. Apparently, the reaction specificity of pyridoxal 5'-phosphate-dependent enzymes is not only achieved by accelerating the specific reaction but also by preventing potential side reactions of the coenzyme substrate adduct.     

Changes in the regiospecificity of aromatic hydroxylation produced by active site engineering in the diiron enzyme toluene 4-monooxygenase

Pseudomonas mendocina KR1 toluene 4-monooxygenase is a multicomponent diiron enzyme. the diiron center is contained in the tmoA polypeptide of teh hydroxylase component [alphabetagamma)2,Mr approximately 212 kDa]. Product distribution studies reveal that the natural isoform is highly specific for para hydroxylation of toluene (kcat approximately 2 s-1 with respect to an alphabetagamma promoter), o-xylene (kcat approximately 0.8 s-1), m-xylene (kcat approximately 0.6 s-1), and other aromatic hydrocarbons. This degree of regioselectivity for methylbenzenes is unmatched by numerous other oxygenase enzymes. However, during the T4MO-catalyzed oxidation of p-xylene (kcat approximately 0.4 s-1), 4-methyl benzyl alcohol is the major product, showing that the enzyme could catalyze either aromatic or benzylic hydroxylation with the appropriate substrate. Site-directed mutagenesis has been used to study the contributions of tmoA active site residues Q141, I180, and F205 to the regiospecificity. Isoforms Q141C and F205I yielded shifts of regiospecificity away from p-cresol formation, with F205I giving an approximately 5-fold increase in the percentage of m-cresol formation relative to that of the natural isoform. The kcat of purified Q141C for toluene oxidation was approximately 0.2 s-1. Isoform Q141C also functioned predominantly as an aromatic ring hydroxylase during the oxidation of p-xylene, in direct contrast to the predominant benzylic hydroxylation observed for the natural isoform, while isoform F205I gave nearly equivalent amounts of benzylic and phenolic products from p-xylene oxidation. Isoform I180F gave no substantial shift in product distributions relativeto the natural isoform for all substrates tested. Upon the basis of a proposed active site model, both Q141 anf F205 are suggested to lie in a hydrophobic region closer to the FeA iron site, while I180 will be closer to FeB. These studies reveal that changes in the hydrophobic region predicted to be nearest to FeA can influence the regiospecificity observed for toluene 4-monooxygenase.     

Gut motility and transit changes in patients receiving long-term methadone maintenance

Chemical modification studies of BamHI endonuclease indicated the importance of the cysteine residue in catalysis [Nath, K. (1981) Arch. Biochem. Biophys, 212, 611-617]. Of the three cysteine residues at positions 34, 54 and 64 in the BamHI endonuclease Cys54 and Cys64 are at the DNA-protein interface. The co-crystal structure of the BamHI-DNA complex, however, does not indicate any role of cysteines either in binding or catalysis. In the context of strong biochemical evidence, Cys54 in BamHI was changed to Ala54 to investigate its role in catalysis. The mutation was carried out by PCR overlap extension, the mutant gene was cloned and characterized by sequencing. The mutant BamHI was expressed and purified to homogeneity and the kinetic parameters (K(M) and kcat) of the wild type and the C54A mutant were determined. The mutation results in up to approximately 40% enhancement of kcat and some increase in K(M). These in vitro results were also supported by in vivo SOS induction assays: the C54A mutant gene under the T7 promoter caused complete lysis in JH139 in absence of T7 RNA polymerase whereas the wild-type gene gave deep blue colonies under the same conditions. The results suggest no direct role of Cys54 in catalysis, but it can influence the catalytic activity through Val57 backbone contact seen in the co-crystal structure.     

Pre-steady-state kinetic analysis of the trichodiene synthase reaction pathway

The pre-steady-state kinetics of the trichodiene synthase reaction were investigated by rapid chemical quench methods. The single-turnover rate was found to be 3.5-3.8 s-1, a rate 40 times faster than the steady-state catalytic rate (kcat = 0.09 s-1) for trichodiene synthase-catalyzed conversion of farnesyl diphosphate (FPP) to trichodiene at 15 degrees C. In a multiturnover experiment, a burst phase (kb = 4.2 s-1) corresponding to the accumulation of trichodiene on the surface of the enzyme was followed by a slower, steady-state release of products (klin = 0.086 s-1) which corresponds to kcat. These results strongly suggest that the release of trichodiene from the enzyme active site is the rate-limiting step in the overall reaction, while the consumption of FPP is the step which limits chemical catalysis at the active site. Single-turnover experiments with trichodiene synthase mutant D101E, for which the steady-state rate constant kcat is 1/3 of that of wild type, revealed that the mutation actually depresses the rate of FPP consumption by a factor of 100. The deuterium isotope effect on the consumption of [1-2H,1,2-14C]FPP was found to be 1.11 +/- 0.06. Single turnover reactions of [1,2-14C]FPP catalyzed by trichodiene synthase were carried out at 4, 15, or 30 degrees C in an effort to provide direct observation of the proposed intermediate nerolidyl diphosphate (NPP). However, no NPP was detected, indicating that the conversion of NPP must be too fast to be observed within the detection limits of the assay. Taken together, these observations suggest that the isomerization of FPP to NPP is the step which limits the rate of chemical catalysis in the trichodiene synthase reaction pathway.     

Site-directed mutagenesis of the active site glutamate in human matrilysin: investigation of its role in catalysis

Glu-198 of human matrilysin is a conserved residue in the matrix metalloproteinases and is considered to play an important role in catalysis by acting as a general base catalyst toward the zinc-bound water molecule, on the basis of mechanistic proposals for other zinc proteases. In the present study, Glu-198 is mutated into Asp, Cys, Gln, and Ala, and the zinc binding properties, kinetic parameters, and pH dependence of each mutant are determined in order to examine the role of Glu-198 in catalysis. The mutations chosen either modify (C and D) or eliminate (A and Q) the general base properties of residue-198. All the mutants bind 2 mol of zinc per mol of enzyme, indicating that Glu-198 is not crucial to the binding of the catalytic zinc to the enzyme. The value of kcat/Km for the E198D mutant is only 4-fold lower than that of wild-type enzyme at the pH optimum of 7.5, while that for the E198C mutant is decreased by 160-fold. The E198Q and E198A enzymes containing the mutations that have eliminated the nucleophilic and acid/base properties of the residue are still active, having lower kcat/Km values of 590- and 1900-fold, respectively. The decrease in activity of all the mutants is essentially due to a decrease in kcat. The kcat/Km values of the mutants as a function of pH display broad bell-shaped curves that are similar to the wild-type enzyme. The acidic pKa value is not greatly affected by the change in the chemical properties of residue-198. The similarity in the pH profiles for the mutant and wild-type enzymes indicates that the ionization of Glu-198 is not responsible for the acidic pKa. Ionization of the zinc-bound water may be responsible for this pKa since the three His ligands and the scaffolding of the matrilysin catalytic zinc site are different from that observed in carboxypeptidase A and would predict a lower pKa for the metal-bound water. If the zinc-bound water is the nucleophile in the reaction, the role of Glu-198 in catalysis may be to stabilize the transition state or act as a general acid catalyst after the rate-determining step.