Identification of a peroxide-sensitive redox switch at the CXXC motif in the human mitochondrial branched chain aminotransferase

The human mitochondrial branched chain aminotransferase isoenzyme (hBCATm) must be stored in a reducing environment to remain active. Oxidation or labeling of hBCATm with sulfhydryl reagents results in enzyme inhibition. In this study, we investigated both the structural and biochemical basis for the sensitivity of hBCATm to these reagents. In its native form, hBCATm has two reactive cysteine residues which were identified as Cys315 and Cys318 using iodinated beta-(4-hydroxyphenyl)ethyl maleimide. These are located in the large domain of the homodimer, about 10 A from the active site. The crystal structures show evidence for a thiol-thiolate hydrogen bond between Cys315 and Cys318. Under oxidizing conditions, these cysteine residues can reasonably form a disulfide bond because of the short distance between the sulfur atoms (3.09-3.46 A), requiring only a decrease of 1.1-1.5 A. In addition to Cys315 playing a structural role by anchoring Tyr173, which in the ketimine form increases access to the active site, our evidence indicates that these cysteine residues act as a redox switch in hBCATm. Electrospray ionization mass spectrometry analysis and UV-Vis spectroscopic studies of 5,5'-dithiobis(2-nitrobenzoic acid) labeled hBCATm showed that during labeling, an intrasubunit disulfide bond was formed in a significant portion of the protein. Furthermore, it was established that reaction of hBCATm with H2O2 abolished its activity and resulted in the formation of an intrasubunit disulfide bond between Cys315 and Cys318. Addition of dithiothreitol completely reversed the oxidation and restored activity. Therefore, the results demonstrate that there is redox-linked regulation of hBCATm activity by a peroxide sensitive CXXC center. Future studies will determine if this center has an in vivo role in the regulation of branched chain amino acid metabolism.     

Human mitochondrial branched chain aminotransferase isozyme: structural role of the CXXC center in catalysis

Mammalian branched chain aminotransferases (BCATs) have a unique CXXC center. Kinetic and structural studies of three CXXC center mutants (C315A, C318A, and C315A/C318A) of human mitochondrial (hBCATm) isozyme and the oxidized hBCATm enzyme (hBCATm-Ox) have been used to elucidate the role of this center in hBCATm catalysis. X-ray crystallography revealed that the CXXC motif, through its network of hydrogen bonds, plays a crucial role in orienting the substrate optimally for catalysis. In all structures, there were changes in the structure of the beta-turn preceding the CXXC motif when compared with wild type protein. The N-terminal loop between residues 15 and 32 is flexible in the oxidized and mutant enzymes, the disorder greater in the oxidized protein. Disordering of the N-terminal loop disrupts the integrity of the side chain binding pocket, particularly for the branched chain side chain, less so for the dicarboxylate substrate side chain. The kinetic studies of the mutant and oxidized enzymes support the structural analysis. The kinetic results showed that the predominant effect of oxidation was on the second half-reaction rather than the first half-reaction. The oxidized enzyme was completely inactive, whereas the mutants showed limited activity. Model building of the second half-reaction substrate alpha-ketoisocaproate in the pyridoxamine 5'-phosphate-hBCATm structure suggests that disruption of the CXXC center results in altered substrate orientation and deprotonation of the amino group of pyridoxamine 5'-phosphate, which inhibits catalysis.     

Human mitochondrial branched chain aminotransferase: structural basis for substrate specificity and role of redox active cysteines

Crystal structures of the fold type IV pyridoxal phosphate (PLP)-dependent human mitochondrial branched chain aminotransferase (hBCATm) reaction intermediates have provided a structural explanation for the kinetically determined substrate specificity of hBCATm. The isoleucine side chain in the ketimine intermediate occupies a hydrophobic binding pocket that can be defined by three surfaces. Modeling of amino acids on the ketimine structure shows that the side chains of nonsubstrate amino acids such as the aromatic amino acids, alanine, or aspartate either are unable to interact through van der Waals' interactions or have steric clashes. The structural and biochemical basis for the sensitivity of the mammalian BCAT to reducing agents has also been elucidated. Two cysteine residues in hBCATm, Cys315 and Cys318 (CXXC), are part of a redox-controlled mechanism that can regulate hBCATm activity. The residues surrounding Cys315 and Cys318 show considerable sequence conservation in the prokaryotic and eukaryotic BCAT sequences, however, the CXXC motif is found only in the mammalian proteins. The results suggest that the BCAT enzymes may join the list of enzymes that can be regulated by redox status.     

Analysis of coumarin 7-hydroxylation activity of cytochrome P450 2A6 using random mutagenesis

Cytochrome P450 (P450) 2A6 is an important human enzyme involved in the metabolism of many xenobiotic chemicals including coumarin, indole, nicotine, and carcinogenic nitrosamines. A combination of random mutagenesis and high-throughput screening was used in the analysis of P450 2A6, utilizing a fluorescent coumarin 7-hydroxylation assay. The steady-state kinetic parameters (k(cat) and Km) for coumarin 7-hydroxylation by wild-type P450 2A6 and 35 selected mutants were measured and indicated that mutants throughout the coding region can have effects on activity. Five mutants showing decreased catalytic efficiency (k(cat)/Km) were further analyzed for substrate selectivity and binding affinities and showed reduced catalytic activities for 7-methoxycoumarin O-demethylation, tert-butyl methyl ether O-demethylation, and indole 3-hydroxylation. All mutants except one (K476E) showed decreased coumarin binding affinities (and also higher Km values), indicating that this is a major basis for the decreased enzymatic activities. A recent x-ray crystal structure of P450 2A6 bound to coumarin (Yano, J. K., Hsu, M. H., Griffin, K. J., Stout, C. D., and Johnson, E. F. (2005) Nat. Struct. Mol. Biol. 12, 822-823) indicates that the recovered A481T and N297S mutations appear to be close to coumarin, suggesting direct perturbation of substrate interaction. The decreased enzymatic activity of the K476E mutant was associated with decreases both in NADPH oxidation and the reduction rate of the ferric P450 2A6-coumarin complex. The attenuation is caused in part to lower binding affinity for NADPH-P450 reductase, but the K476E mutant did not achieve the wild-type coumarin 7-hydroxylation activity even at high reductase concentrations.     

Differential redox potential between the human cytosolic and mitochondrial branched-chain aminotransferase

The human branched-chain aminotransferase (hBCAT) isoenzymes are CXXC motif redox sensitive homodimers central to glutamate metabolism in the central nervous system. These proteins respond differently to oxidation by H(2)O(2), NO, and S-glutathionylation, suggesting that the redox potential is distinct between isoenzymes. Using various reduced to oxidized glutathione ratios (GSH:GSSG) to alter the redox environment, we demonstrate that hBCATc (cytosolic) has an overall redox potential that is 30 mV lower than hBCATm (mitochondrial). Furthermore, the CXXC motif of hBCATc was estimated to be 80 mV lower, suggesting that hBCATm is more oxidizing in nature. Western blot analysis revealed close correlations between hBCAT S-glutathionylation and the redox status of the assay environment, offering the hBCAT isoenzymes as novel biomarkers for cytosolic and mitochondrial oxidative stress.     

A novel branched-chain amino acid metabolon. Protein-protein interactions in a supramolecular complex

The catabolic pathways of branched-chain amino acids have two common steps. The first step is deamination catalyzed by the vitamin B(6)-dependent branched-chain aminotransferase isozymes (BCATs) to produce branched-chain alpha-keto acids (BCKAs). The second step is oxidative decarboxylation of the BCKAs mediated by the branched-chain alpha-keto acid dehydrogenase enzyme complex (BCKD complex). The BCKD complex is organized around a cubic core consisting of 24 lipoate-bearing dihydrolipoyl transacylase (E2) subunits, associated with the branched-chain alpha-keto acid decarboxylase/dehydrogenase (E1), dihydrolipoamide dehydrogenase (E3), BCKD kinase, and BCKD phosphatase. In this study, we provide evidence that human mitochondrial BCAT (hBCATm) associates with the E1 decarboxylase component of the rat or human BCKD complex with a K(D) of 2.8 microM. NADH dissociates the complex. The E2 and E3 components do not interact with hBCATm. In the presence of hBCATm, k(cat) values for E1-catalyzed decarboxylation of the BCKAs are enhanced 12-fold. Mutations of hBCATm proteins in the catalytically important CXXC center or E1 proteins in the phosphorylation loop residues prevent complex formation, indicating that these regions are important for the interaction between hBCATm and E1. Our results provide evidence for substrate channeling between hBCATm and BCKD complex and formation of a metabolic unit (termed branched-chain amino acid metabolon) that can be influenced by the redox state in mitochondria.     

Studies of the enzymic mechanism of Candida tenuis xylose reductase (AKR 2B5): X-ray structure and catalytic reaction profile for the H113A mutant

Xylose reductase from the yeast Candida tenuis (CtXR) is a family 2 member of the aldo-keto reductase (AKR) superfamily of proteins and enzymes. Active site His-113 is conserved among AKRs, but a unified mechanism of how it affects catalytic activity is outstanding. We have replaced His-113 by alanine using site-directed mutagenesis, determined a 2.2 A structure of H113A mutant bound to NADP(+), and compared catalytic reaction profiles of NADH-dependent reduction of different aldehydes catalyzed by the wild type and the mutant. Deuterium kinetic isotope effects (KIEs) on k(cat) and k(cat)/K(m xylose) show that, relative to the wild type, the hydride transfer rate constant (k(7) approximately 0.16 s(-1)) has decreased about 1000-fold in H113A whereas xylose binding was not strongly affected. No solvent isotope effect was seen on k(cat) and k(cat)/K(m xylose) for H113A, suggesting that proton transfer has not become rate-limiting as a result of the mutation. The pH profiles of log(k(cat)/K(m xylose)) for the wild type and H113A decreased above apparent pK(a) values of 8.85 and 7.63, respectively. The DeltapK(a) of -1.2 pH units likely reflects a proximally disruptive character of the mutation, affecting the position of Asp-50. A steady-state kinetic analysis for H113A-catalyzed reduction of a homologous series of meta-substituted benzaldehyde derivatives was carried out, and quantitative structure-reactivity correlations were used to factor the observed kinetic substituent effect on k(cat) and k(cat)/K(m aldehyde) into an electronic effect and bonding effects (which are lacking in the wild type). Using the Hammett sigma scale, electronic parameter coefficients (rho) of +0.64 (k(cat)) and +0.78 (k(cat)/K(m aldehyde)) were calculated and clearly differ from rho(k(cat)/K(aldehyde)) and rho(k(cat)) values of +1.67 and approximately 0.0, respectively, for the wild-type enzyme. Hydride transfer rate constants of H113A, calculated from kinetic parameters and KIE data, display a substituent dependence not seen in the corresponding wild-type enzyme rate constants. An enzymic mechanism is proposed in which His-113, through a hydrogen bond from Nepsilon2 to aldehyde O1, assists in catalysis by optimizing the C=O bond charge separation and orbital alignment in the ternary complex.     

Catalytic mechanism of thiol peroxidase from Escherichia coli. Sulfenic acid formation and overoxidation of essential CYS61

Escherichia coli thiol peroxidase (Tpx, p20, scavengase) is part of an oxidative stress defense system that uses reducing equivalents from thioredoxin (Trx1) and thioredoxin reductase to reduce alkyl hydroperoxides. Tpx contains three Cys residues, Cys(95), Cys(82), and Cys(61), and the latter residue aligns with the N-terminal active site Cys of other peroxidases in the peroxiredoxin family. To identify the catalytically important Cys, we have cloned and purified Tpx and four mutants (C61S, C82S, C95S, and C82S,C95S). In rapid reaction kinetic experiments measuring steady-state turnover, C61S is inactive, C95S retains partial activity, and the C82S mutation only slightly affects reaction rates. Furthermore, a sulfenic acid intermediate at Cys(61) generated by cumene hydroperoxide (CHP) treatment was detected in UV-visible spectra of 4-nitrobenzo-2-oxa-1,3-diazole-labeled C82S,C95S, confirming the identity of Cys(61) as the peroxidatic center. In stopped-flow kinetic studies, Tpx and Trx1 form a Michaelis complex during turnover with a catalytic efficiency of 3.0 x 10(6) m(-1) s(-1), and the low K(m) (9.0 microm) of Tpx for CHP demonstrates substrate specificity toward alkyl hydroperoxides over H(2)O(2) (K(m) > 1.7 mm). Rapid inactivation of Tpx due to Cys(61) overoxidation is observed during turnover with CHP and a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid, but not H(2)O(2). Unlike most other 2-Cys peroxiredoxins, which operate by an intersubunit disulfide mechanism, Tpx contains a redox-active intrasubunit disulfide bond yet is homodimeric in solution.     

Catalytic properties of dihydroorotate dehydrogenase from Saccharomyces cerevisiae: studies on pH, alternate substrates, and inhibitors

Yeast dihydroorotate dehydrogenase (DHOD) was purified 2800-fold to homogeneity from its natural source. Its sequence is 70% identical to that of the Lactococcus lactis DHOD (family IA) and the two active sites are nearly the same. Incubations of the yeast DHOD with dideuterodihydroorotate (deuterated in the positions eliminated in the dehydrogenation) as the donor and [14C]orotate as the acceptor revealed that the C5 deuteron exchanged with H2O solvent at a rate equal to the 14C exchange rate, whereas the C6 deuteron was infrequently exchanged with H2O solvent, thus indicating that the C6 deuteron of the dihydroorotate is sticky on the flavin cofactor. The pH dependencies of the steady-state parameters (k(cat) and k(cat)/Km) are similar, indicating that k(cat)/Km reports the productive binding of substrate, and the parameters are dependent on the donor-acceptor pair. The lower pKa values for k(cat) and k(cat)/Km observed for substrate dihydroorotate (around 6) in comparison to the values determined for dihydrooxonate (around 8) suggest that the C5 pro S hydrogen atom of dihydroorotate (but not the analogous hydrogen of dihydrooxonate), which is removed in the dehydrogenation, assists in lowering the pKa of the active site base (Cys133). The pH dependencies of the kinetic isotope effects on steady-state parameters observed for the dideuterated dihydroorotate are consistent with the dehydrogenation of substrate being rate limiting at low pH values, with a pKa value approximating that assigned to Cys133. Electron acceptors with dihydroorotate as donor were preferred in the following order: ferricyanide (1), DCPIP (0.54), Qo (0.28), fumarate (0.15), and O2 (0.035). Orotate inhibition profiles versus varied concentrations of dihydroorotate with ferricyanide or O2 as acceptors suggest that both orotate and dihydroorotate have significant affinities for the reduced and oxidized forms of the enzyme.     

The CXXC motif: imperatives for the formation of native disulfide bonds in the cell.

The rapid formation of native disulfide bonds in cellular proteins is necessary for the efficient use of cellular resources. This process is catalyzed in vitro by protein disulfide isomerase (PDI), with the PDI1 gene being essential for the viability of Saccharomyces cerevisiae. PDI is a member of the thioredoxin (Trx) family of proteins, which have the active-site motif CXXC. PDI contains two Trx domains as well as two domains unrelated to the Trx family. We find that the gene encoding Escherichia coli Trx is unable to complement PDI1 null mutants of S.cerevisiae. Yet, Trx can replace PDI if it is mutated to have a CXXC motif with a disulfide bond of high reduction potential and a thiol group of low pKa. Thus, an enzymic thiolate is both necessary and sufficient for the formation of native disulfide bonds in the cell.     

Roles of the Redox-Active Disulfide and Histidine Residues Forming a Catalytic Dyad in Reactions Catalyzed by 2-Ketopropyl Coenzyme M Oxidoreductase/Carboxylase

NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC), an atypical member of the disulfide oxidoreductase (DSOR) family of enzymes, catalyzes the reductive cleavage and carboxylation of 2-ketopropyl-coenzyme M [2-(2-ketopropylthio)ethanesulfonate; 2-KPC] to form acetoacetate and coenzyme M (CoM) in the bacterial pathway of propylene metabolism. Structural studies of 2-KPCC from Xanthobacter autotrophicus strain Py2 have revealed a distinctive active-site architecture that includes a putative catalytic triad consisting of two histidine residues that are hydrogen bonded to an ordered water molecule proposed to stabilize enolacetone formed from dithiol-mediated 2-KPC thioether bond cleavage. Site-directed mutants of 2-KPCC were constructed to test the tenets of the mechanism proposed from studies of the native enzyme. Mutagenesis of the interchange thiol of 2-KPCC (C82A) abolished all redox-dependent reactions of 2-KPCC (2-KPC carboxylation or protonation). The air-oxidized C82A mutant, as well as wild-type 2-KPCC, exhibited the characteristic charge transfer absorbance seen in site-directed variants of other DSOR enzymes but with a pK_a value for C87 (8.8) four units higher (i.e., four orders of magnitude less acidic) than that for the flavin thiol of canonical DSOR enzymes. The same higher pK_a value was observed in native 2-KPCC when the interchange thiol was alkylated by the CoM analog 2-bromoethanesulfonate. Mutagenesis of the flavin thiol (C87A) also resulted in an inactive enzyme for steady-state redox-dependent reactions, but this variant catalyzed a single-turnover reaction producing a 0.8:1 ratio of product to enzyme. Mutagenesis of the histidine proximal to the ordered water (H137A) led to nearly complete loss of redox-dependent 2-KPCC reactions, while mutagenesis of the distal histidine (H84A) reduced these activities by 58 to 76%. A redox-independent reaction of 2-KPCC (acetoacetate decarboxylation) was not decreased for any of the aforementioned site-directed mutants. We interpreted and rationalized these results in terms of a mechanism of catalysis for 2-KPCC employing a unique hydrophobic active-site architecture promoting thioether bond cleavage and enolacetone formation not seen for other DSOR enzymes.     

Catalytic acid-base groups in yeast pyruvate decarboxylase. 1. Site-directed mutagenesis and steady-state kinetic studies on the enzyme with the D28A, H114F, H115F, and E477Q substitutions.

The roles of four of the active center groups with potential acid-base properties in the region of pH optimum of pyruvate decarboxylase from Saccharomyces cerevisiae have been studied with the substitutions Asp28Ala, His114Phe, His115Phe, and Glu477Gln, introduced by site-directed mutagenesis methods. The steady-state kinetic constants were determined in the pH range of activity for the enzyme. The substitutions result in large changes in k(cat) and k(cat)/S(0.5) (and related terms), indicating that all four groups have a role in transition state stabilization. Furthermore, these results also imply that all four are involved in some manner in stabilizing the rate-limiting transition state(s) both at low substrate (steps starting with substrate binding and culminating in decarboxylation) and at high substrate concentration (steps beginning with decarboxylation and culminating in product release). With the exception of some modest effects, the shapes of neither the bell-shaped k(cat)/S(0.5)-pH (and related functions) plots nor the k(cat)-pH plots are changed by the substitutions. Yet, the fractional activity still remaining after substitutions virtually rules out any of the four residues as being directly responsible for initiating the catalytic process by ionizing the C2H. There is no effect on the C2 H/D exchange rate exhibited by the D28A and E477Q substitutions. These results strongly imply that the base-induced deprotonation at C2 is carried out by the only remaining base, the iminopyrimidine tautomer of the coenzyme, via intramolecular proton abstraction. The first product is released as CO(2) rather than HCO(3)(-) by both wild-type and E477Q and D28A variants, ruling out several mechanistic alternatives.     

Thioredoxin-2 but not thioredoxin-1 is a substrate of thioredoxin peroxidase-1 from Drosophila melanogaster: isolation and characterization of a second thioredoxin in D. Melanogaster and evidence for distinct biological functions of Trx-1 and Trx-2

As Drosophila melanogaster does not contain glutathione reductase, the thioredoxin system has a key function for glutathione disulfide reduction in insects (Kanzok, S. M., Fechner, A., Bauer, H., Ulschmid, J. K., Müller, H. M., Botella-Munoz, J., Schneuwly, S., Schirmer, R. H., and Becker, K. (2001) Science 291, 643-646). In view of these unique conditions, the protein systems participating in peroxide metabolism and in redox signaling are of special interest. The genes for a second thioredoxin (DmTrx-2) and a thioredoxin peroxidase (DmTPx-1) were cloned and expressed, and the proteins were characterized. In its disulfide form, the 13-kDa protein thioredoxin-2 is a substrate of thioredoxin reductase-1 (K(m) = 5.2 microm, k(cat) = 14.5 s(-1)) and in its dithiol form, an electron donor for TPx-1 (K(m) = 9 microm, k(cat) = 5.4 s(-1)). DmTrx-2 is capable of reducing glutathione disulfide with a second order rate constant of 170 m(-1) s(-1) at pH 7.4 and 25 degrees C. Western blot analysis indicated that this thioredoxin represents up to 1% of the extractable protein of D. melanogaster Schneider cells or whole fruit flies. Recombinant thioredoxin peroxidase-1 (subunit molecular mass = 23 kDa) was found to be a decameric protein that can efficiently use Trx-2 but not Trx-1 as a reducing substrate. The new electron pathway found in D. melanogaster is also representative for insects that serve as vectors of disease. As a first step we have cloned and functionally expressed the gene that is the orthologue of DmTrx-2 in the malaria mosquito Anopheles gambiae.     

The catalytic mechanism of carbonic anhydrase. Hydrogen-isotope effects on the kinetic parameters of the human C isoenzyme.

1. The steady-state kinetics of the interconversion of CO2 and HCO3 catalyzed by human carbonic anhydrase C was studied using 1H2O and 2H2O as solvents. The pH-independent parts of the parameters k(cat) and Km are 3-4 times larger in 1H2O than in 2H2O for both directions of the reaction, while the ratios k(cat)/Km show much smaller isotope effects. With either CO2 or HCO3 as substrate the major pH dependence is observed in k(cat), while Km appears independent of pH. The pKa value characterizing the pH-rate profiles is approximately 0.5 unit larger in 2H2O than in 1H2O. 2. The hydrolysis of p-nitrophenyl acetate catalyzed by human carbonic anhudrase C is approximately 35% faster in 2H2O than in 1H2O. In both solvents the pKa values of the pH-rate profiles are similar to those observed for the CO2-HCO3 interconversion. 3. It is tentatively proposed that the rate-limiting step at saturating concentrations of CO2 or HCO3 is an intramolecular proton transfer between two ionizing groups in the active site. It cannot be decided whether the transformation between enzyme-bound CO2 and HCO3 involves a proton trnasfer or not.     

Structural determinants for branched-chain aminotransferase isozyme-specific inhibition by the anticonvulsant drug gabapentin

This study presents the first three-dimensional structures of human cytosolic branched-chain aminotransferase (hBCATc) isozyme complexed with the neuroactive drug gabapentin, the hBCATc Michaelis complex with the substrate analog, 4-methylvalerate, and the mitochondrial isozyme (hBCATm) complexed with gabapentin. The branched-chain aminotransferases (BCAT) reversibly catalyze transamination of the essential branched-chain amino acids (leucine, isoleucine, valine) to alpha-ketoglutarate to form the respective branched-chain alpha-keto acids and glutamate. The cytosolic isozyme is the predominant BCAT found in the nervous system, and only hBCATc is inhibited by gabapentin. Pre-steady state kinetics show that 1.3 mm gabapentin can completely inhibit the binding of leucine to reduced hBCATc, whereas 65.4 mm gabapentin is required to inhibit leucine binding to hBCATm. Structural analysis shows that the bulky gabapentin is enclosed in the active-site cavity by the shift of a flexible loop that enlarges the active-site cavity. The specificity of gabapentin for the cytosolic isozyme is ascribed at least in part to the location of the interdomain loop and the relative orientation between the small and large domain which is different from these relationships in the mitochondrial isozyme. Both isozymes contain a CXXC center and form a disulfide bond under oxidizing conditions. The structure of reduced hBCATc was obtained by soaking the oxidized hBCATc crystals with dithiothreitol. The close similarity in active-site structures between cytosolic enzyme complexes in the oxidized and reduced states is consistent with the small effect of oxidation on pre-steady state kinetics of the hBCATc first half-reaction. However, these kinetic data do not explain the inactivation of hBCATm by oxidation of the CXXC center. The structural data suggest that there is a larger effect of oxidation on the interdomain loop and residues surrounding the CXXC center in hBCATm than in hBCATc.     

Stereoselective detoxification of chiral sarin and soman analogues by phosphotriesterase

The catalytic activity of the bacterial phosphotriesterase (PTE) toward a series of chiral analogues of the chemical warfare agents sarin and soman was measured. Chemical procedures were developed for the chiral syntheses of the S(P)- and R(P)-enantiomers of O-isopropyl p-nitrophenyl methylphosphonate (sarin analogue) in high enantiomeric excess. The R(P)-enantiomer of the sarin analogue (k(cat)=2600 s(-1)) was the preferred substrate for the wild-type PTE relative to the corresponding S(P)-enantiomer (k(cat)=290 s(-1)). The observed stereoselectivity was reversed using the PTE mutant, I106A/F132A/H254Y where the k(cat) values for the R(P)- and S(P)-enantiomers were 410 and 4200 s(-1), respectively. A chemo-enzymatic procedure was developed for the chiral synthesis of the four stereoisomers of O-pinacolyl p-nitrophenyl methylphosphonate (soman analogue) with high diastereomeric excess. The R(P)R(C)-stereoisomer of the soman analogue was the preferred substrate for PTE. The k(cat) values for the soman analogues were measured as follows: R(P)R(C,) 48 s(-1); R(P)S(C), 4.8 s(-1); S(P)R(C), 0.3 s(-1), and S(P)S(C), 0.04 s(-1). With the I106A/F132A/H254Y mutant of PTE the stereoselectivity toward the chiral phosphorus center was reversed. With the triple mutant the k(cat) values for the soman analogues were found to be as follows: R(P)R(C,) 0.3 s(-1); R(P)S(C), 0.3 s(-1); S(P)R(C), 11s(-1), and S(P)S(C), 2.1 s(-1). Prior investigations have demonstrated that the S(P)-enantiomers of sarin and soman are significantly more toxic than the R(P)-enantiomers. This investigation has demonstrated that mutants of the wild-type PTE can be readily constructed with enhanced catalytic activities toward the most toxic stereoisomers of sarin and soman.     

Staphylococcus aureus sortase transpeptidase SrtA: insight into the kinetic mechanism and evidence for a reverse protonation catalytic mechanism

The Staphylococcus aureus transpeptidase SrtA catalyzes the covalent attachment of LPXTG-containing virulence and colonization-associated proteins to cell-wall peptidoglycan in Gram-positive bacteria. Recent structural characterizations of staphylococcal SrtA, and related transpeptidases SrtB from S. aureus and Bacillus anthracis, provide many details regarding the active site environment, yet raise questions with regard to the nature of catalysis and active site cysteine thiol activation. Here we re-evaluate the kinetic mechanism of SrtA and shed light on aspects of its catalytic mechanism. Using steady-state, pre-steady-state, bisubstrate kinetic studies, and high-resolution electrospray mass spectrometry, revised steady-state kinetic parameters and a ping-pong hydrolytic shunt kinetic mechanism were determined for recombinant SrtA. The pH dependencies of kinetic parameters k(cat)/K(m) and k(cat) for the substrate Abz-LPETG-Dap(Dnp)-NH(2) were bell-shaped with pK(a) values of 6.3 +/- 0.2 and 9.4 +/- 0.2 for k(cat) and 6.2 +/- 0.2 and 9.4 +/- 0.2 for k(cat)/K(m). Solvent isotope effect (SIE) measurements revealed inverse behavior, with a (D)2(O)k(cat) of 0.89 +/- 0.01 and a (D)2(O)(k(cat)/K(m)) of 0.57 +/- 0.03 reflecting an equilibrium SIE. In addition, SIE measurements strongly implicated Cys184 participation in the isotope-sensitive rate-determining chemical step when considered in conjunction with an inverse linear proton inventory for k(cat). Last, the pH dependence of SrtA inactivation by iodoacetamide revealed a single ionization for inactivation. These studies collectively provide compelling evidence for a reverse protonation mechanism where a small fraction (ca. 0.06%) of SrtA is competent for catalysis at physiological pH, yet is highly active with an estimated k(cat)/K(m) of >10(5) M(-)(1) s(-)(1).     

Active site serine promotes stabilization of the reactive glutathione thiolate in rat glutathione transferase T2-2. Evidence against proposed sulfatase activity of the corresponding human enzyme

Ser(11) in rat glutathione transferase T2-2 is important for stabilization of the reactive enzyme-bound glutathione thiolate in the reaction with 1-menaphthyl sulfate. The S11A mutation increased the pK(a) value for the pH dependence of the rate constant for pre-steady-state product formation, from 5.7 to 7.9. This pH dependence is proposed to reflect titration of enzyme-bound glutathione thiol. Further, the mutation lowered the k(cat) value but not because of the impaired stabilization of the glutathione thiolate. In fact, several steps on the reaction pathway were affected by the S11A mutation, and the cause of the decreased k(cat) for the mutant was found to be a slower product release. The data presented here contradict the hypothesis that glutathione transferase T2-2 could act as a sulfatase that is not dependent on Ser(11) for the catalytic activity, as proposed for the corresponding human enzyme (Tan, K.-L., Chelvanayagam, G., Parker, M. W., and Board, P. G. (1996) Biochem. J. 319, 315-321; Rossjohn, J., McKinstry, W. J., Oakley, A. J., Verger, D., Flanagan, J., Chelvanayagam, G., Tan, K.-L., Board, P. G., and Parker, M. W. (1998) Structure 6, 309-322). On the contrary, Ser(11) governs both chemical and physical steps of the catalyzed reaction.     

The roles of two amino acid residues in the active site of L-lactate monooxygenase. Mutation of arginine 187 to methionine and histidine 240 to glutamine.

Lactate monooxygenase (LMO) catalyzes the conversion of L-lactate to acetate, CO(2), and water with the incorporation of molecular oxygen. Arginine 187 of LMO is highly conserved within the family of L-alpha-hydroxyacid oxidizing enzymes (Lê, K. H. D., and Lederer, F. (1991) J. Biol. Chem. 266, 20877-20881). By comparison with the equivalent residue in flavocytochrome b(2) from Saccharomyces cerevisiae (Pike, A. D., Chapman, S. K, Manson, F. D. C,. Reid, G. A. , Gondry, M., and Lederer, F. (1996) in Flavins and Flavoproteins (Stevenson, K. J., Massey, V., and Williams, C. H., Jr., eds) pp. 571-574, University of Calgary Press, Calgary, AB, Canada), arginine 187 might be expected to have an important role in catalytic efficiency and substrate binding in LMO. Histidine 240 is predicted to be close to the substrate binding site of LMO, although it is not conserved within the enzyme family. Arginine 187 has been replaced with methionine (R187M), and histidine 240 has been replaced with glutamine (H240Q). L-Lactate oxidation by R187M is very slow. The binding of L-lactate to the mutant enzyme appears to be very weak, as is the binding of oxalate, a transition state analogue. The binding of pyruvate to the reduced enzyme is also very weak, resulting in complete uncoupling of enzyme turnover, with H(2)O(2) and pyruvate as the final products. In addition, anionic forms of the flavin are unstable. The K(d) for sulfite is increased nearly 400-fold by this mutation. The semiquinone form of R187M is also thermodynamically unstable, although the overall midpoint potential for the two-electron reduction of R187M is only 34 mV lower than for the wild-type enzyme. H240Q more closely resembles the wild-type enzyme. The steady-state activity of H240Q is completely coupled. The k(cat) is similar to that for the wild-type enzyme.     

Conformational unfolding studies of three-disulfide mutants of bovine pancreatic ribonuclease A and the coupling of proline isomerization to disulfide redox reactions

The equilibrium stability and conformational unfolding kinetics of the [C40A, C95A] and [C65S, C72S] mutants of bovine pancreatic ribonuclease A (RNase A) have been studied. These mutants are analogues of two nativelike intermediates, des[40-95] and des[65-72], whose formation is rate-limiting for oxidative folding and reductive unfolding at 25 degrees C and pH 8.0. Upon addition of guanidine hydrochloride, both mutants exhibit a fast conformational unfolding phase when monitored by absorbance and fluorescence, as well as a slow phase detected only by fluorescence which corresponds to the isomerizations of Pro93 and Pro114. The amplitudes of the slow phase indicate that the two prolines, Pro93 and Pro114, are fully cis in the folded state of the mutants and furthermore that the 40-95 disulfide bond is not responsible for the quenching of Tyr92 fluorescence observed in the slow unfolding phase, contrary to an earlier proposal [Rehage, A., and Schmid, F. X. (1982) Biochemistry 21, 1499-1505]. The ratio of the kinetic unfolding m value to the equilibrium m value indicates that the transition state for conformational unfolding in the mutants exposes little solvent-accessible area, as in the wild-type protein, indicating that the unfolding pathway is not dramatically altered by the reduction of the 40-95 or 65-72 disulfide bond. The stabilities of the folded mutants are compared to that of wild-type RNase A. These stabilities indicate that the reduction of des[40-95] to the 2S species is rate-limited by global conformational unfolding, whereas that of des[65-72] is rate-limited by local conformational unfolding. The isomerization of Pro93 may be rate-limiting for the reduction of the 40-95 disulfide bond in the native protein and in the des[65-72] intermediate.