Remarkable aliphatic hydroxylation by the diiron enzyme toluene 4-monooxygenase in reactions with radical or cation diagnostic probes norcarane, 1,1-dimethylcyclopropane, and 1,1-diethylcyclopropane

Toluene 4-monooxygenase (T4MO) catalyzes the hydroxylation of toluene to yield 96% p-cresol. This diiron enzyme complex was used to oxidize norcarane (bicyclo[4.1.0]heptane), 1,1-dimethylcyclopropane, and 1,1-diethylcyclopropane, substrate analogues that can undergo diagnostic reactions upon the production of transient radical or cationic intermediates. Norcarane closely matches the shape and volume of the natural substrate toluene. Reaction of isoforms of the hydroxylase component of T4MO (T4moH) with different regiospecificities for toluene hydroxylation (k(cat) approximately 1.9-2.3 s(-)(1) and coupling efficiency approximately 81-96%) revealed similar catalytic parameters for norcarane oxidation (k(cat) approximately 0.3-0.5 s(-)(1) and coupling efficiency approximately 72%). The products included variable amounts of the un-rearranged isomeric norcaranols and cyclohex-2-enyl methanol, a product attributed to rearrangement of a radical oxidation intermediate. A ring-expansion product derived from the norcaranyl C-2 cation, cyclohept-3-enol, was not produced by either the natural enzyme or any of the T4moH isoforms tested. Comparative studies of 1,1-dimethylcyclopropane and 1,1-diethylcyclopropane, diagnostic substrates with differences in size and with approximately 50-fold slower k(cat) values, gave products consistent with both radical rearrangement and cation ring expansion. Examination of the isotopic enrichment of the incorporated O-atoms for all products revealed high-fidelity incorporation of an O-atom from O(2) in the un-rearranged and radical-rearranged products, while the O-atom found in the cation ring-expansion products was predominantly obtained by reaction with H(2)O. The results show a divergence of radical and cation pathways for T4moH-mediated hydroxylation that can be dissected by diagnostic substrate probe rearrangements and by changes in the source of oxygen used for substrate oxygenation.     

Mechanistic studies with 2-C-methyl-D-erythritol 4-phosphate synthase from Escherichia coli

The mechanism of the reaction catalyzed by 2-C-methyl-d-erythritol 4-phosphate (MEP) synthase from Escherichia coli has been studied by steady-state and single-turnover kinetic experiments for the 1-deoxy-d-xylulose 5-phosphoric acid (DXP) analogues, 1,1,1-trifluoro-1-deoxy-d-xylulose 5-phosphoric acid (CF(3)-DXP), 1,1-difluoro-1-deoxy-d-xylulose 5-phosphoric acid (CF(2)-DXP), 1-fluoro-1-deoxy-d-xylulose 5-phosphoric acid (CF-DXP), and 1,2-dideoxy-d-hexulose 6-phosphate (Et-DXP). CF(3)-DXP, CF(2)-DXP, and Et-DXP were poor inhibitors, most likely because of the increase in steric bulk at C1 of DXP. The three analogues were also poor substrates for the enzyme. In contrast, CF-DXP was a good substrate (k(cat)(CF)(-)(DXP) = 37 +/- 2 s(-)(1), K(m)(CF)(-)(DXP) = 227 +/- 25 microM) for MEP synthase when compared to DXP (k(cat)(DXP) = 29 +/- 1 s(-)(1), K(m)(DXP) = 45 +/- 4 microM). A primary deuterium isotope effect was observed under single-turnover conditions when CF-DXP was incubated with 4S-[(2)H]NADPH ((H)k/(D)k = 1.34 +/-0.01), whereas no isotope effect was observed upon incubation with DXP and 4S-[(2)H]NADPH ((H)k/(D)k = 1.02 +/- 0.02). The reaction did not exhibit burst kinetics for either substrate, indicating that product release is not rate-limiting. These studies suggest that positive charge does not develop at C2 of DXP during catalysis. In addition, the isotope effect with CF-DXP and 4S-[(2)H]NADPH but not DXP indicates that the rearrangement step, which precedes hydride transfer, is rate-limiting for DXP but becomes partially rate-limiting for CF-DXP. Thus, rearrangement appears to be enhanced by substitution of a hydrogen atom in the methyl group of DXP by fluorine. These observations are consistent with a retro-aldol/aldol mechanism for the rearrangement during conversion of DXP to MEP.     

Negative co-operativity in glutamate dehydrogenase. Involvement of the 2-position in glutamate in the induction of conformational changes.

The 2-position substituent on substrates or substrate analogues for glutamate dehydrogenase is shown to be intimately involved in the induction of conformational changes between subunits in the hexamer by coenzyme. These conformational changes are associated with the negative co-operativity exhibited by this enzyme. 2-Oxoglutarate and L-2-hydroxyglutarate induce indications of co-operativity similar to those induced by the substrate of oxidative deamination, glutamate, in kinetic studies. Glutarate (2-position CH2) does not. A comparison of the effects of L-2-hydroxyglutarate and D-2-hydroxyglutarate or D-glutamate indicates that the 2-position substituent must be in the L-configuration for these conformational changes to be triggered. In addition, glutarate and L-glutamate in ternary enzyme-NAD(P)H-substrate complexes induce very different coenzyme fluorescence properties, showing that glutamate induces a different conformation of the enzyme-coenzyme complex from that induced by glutarate. Although glutamate and glutarate both tighten the binding of reduced coenzyme to the active site, the effect is much greater with glutamate, and the binding is described by two dissociation constants when glutamate is present. The data suggest that the two carboxy groups on the substrate are required to allow synergistic binding of coenzyme and substrate to the active site, but that interactions between the 2-position on the substrate and the enzyme trigger the conformational changes that result in subunit-subunit interactions and in the catalytic co-operativity exhibited by this enzyme.     

Mechanistic studies on thiamin phosphate synthase: evidence for a dissociative mechanism

Thiamin phosphate synthase catalyzes the coupling of 4-methyl-5-(beta-hydroxyethyl)thiazole phosphate (Thz-P) and 4-amino-5-(hydroxymethyl)-2-methylpyrimidine pyrophosphate (HMP-PP) to give thiamin phosphate. In this paper, we demonstrate that 4-amino-5-(hydroxymethyl)-2-(trifluoromethyl)pyrimidine pyrophosphate (CF(3)-HMP-PP) is a very poor substrate [k(cat)(CH(3)) > 7800k(cat)(CF(3))] and that 4-amino-5-(hydroxymethyl)-2-methoxypyrimidine pyrophosphate (CH(3)O-HMP-PP) is a good substrate [k(cat)(OCH(3)) > 2.8k(cat)(CH(3))] for the enzyme. We also demonstrate that the enzyme catalyzes positional isotope exchange. These observations are consistent with a dissociative mechanism (S(N)1 like) for thiamin phosphate synthase in which the pyrimidine pyrophosphate dissociates to give a reactive pyrimidine intermediate which is then trapped by the thiazole moiety.     

Protection of radical intermediates at the active site of adenosylcobalamin-dependent methylmalonyl-CoA mutase

Adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the interconversion of methylmalonyl-CoA and succinyl-CoA via radical intermediates generated by substrate-induced homolysis of the coenzyme carbon-cobalt bond. From the structure of methylmalonyl-CoA mutase it is evident that the deeply buried active site is completely shielded from solvent with only a few polar contacts made between the protein and the substrate. Site-directed mutants of amino acid His244, a residue close to the inferred site of radical chemistry, were engineered to investigate its role in catalysis. Two mutants, His244Ala and His244Gln, were characterized using kinetic and spectroscopic techniques. These results confirmed that His244 is not an essential residue. However, compared with that of the wild type, k(cat) was lowered by 10(2)- and 10(3)-fold for the His244Gln and His244Ala mutants, respectively, while the K(m) for succinyl-CoA was essentially unchanged in both cases. The primary kinetic tritium isotope effect (k(H)/k(T)) for the His244Gln mutant was 1.5 +/- 0.3, and tritium partitioning was now found to be dependent on the substrate used to initiate the reaction, indicating that the rearrangement of the substrate radical to the product radical was extremely slow. The His244Ala mutant underwent inactivation under aerobic conditions at a rate between 1 and 10% of the initial rate of turnover. The crystal structure of the His244Ala mutant, determined at 2.6 A resolution, indicated that the mutant enzyme is unaltered except for a cavity in the active site which is occupied by an ordered water molecule. Molecular oxygen reaching this cavity may lead directly to inactivation. These results indicate that His244 assists directly in the unusual carbon skeleton rearrangement and that alterations in this residue substantially lower the protection of reactive radical intermediates during catalysis.     

Proton transfer from histidine 244 may facilitate the 1,2 rearrangement reaction in coenzyme B(12)-dependent methylmalonyl-CoA mutase.

Methylmalonyl-CoA mutase is an adenosylcobalamin-dependent enzyme that catalyzes the 1,2 rearrangement of methylmalonyl-CoA to succinyl-CoA. This reaction results in the interchange of a carbonyl-CoA group and a hydrogen atom on vicinal carbons. The crystal structure of the enzyme reveals the presence of an aromatic cluster of residues in the active site that includes His-244, Tyr-243, and Tyr-89 in the large subunit. Of these, His-244 is within hydrogen bonding distance to the carbonyl oxygen of the carbonyl-CoA moiety of the substrate. The location of these aromatic residues suggests a possible role for them in catalysis either in radical stabilization and/or by direct participation in one or more steps in the reaction. The mechanism by which the initially formed substrate radical isomerizes to the product radical during the rearrangement of methylmalonyl-CoA to succinyl-CoA is unknown. Ab initio molecular orbital theory calculations predict that partial proton transfer can contribute significantly to the lowering of the barrier for the rearrangement reaction. In this study, we report the kinetic characterization of the H244G mutant, which results in an acute sensitivity of the enzyme to oxygen, indicating the important role of this residue in radical stabilization. Mutation of His-244 leads to an approximately 300-fold lowering in the catalytic efficiency of the enzyme and loss of one of the two titratable pK(a) values that govern the activity of the wild type enzyme. These data suggest that protonation of His-244 increases the reaction rate in wild type enzyme and provides experimental support for ab initio molecular orbital theory calculations that predict rate enhancement of the rearrangement reaction by the interaction of the migrating group with a general acid. However, the magnitude of the rate enhancement is significantly lower than that predicted by the theoretical studies.     

The mechanism of adenosylcobalamin-dependent rearrangements

Adenosylcobalamin (AdoCbl)-dependent rearrangements are a group of reactions with no obvious precedents in organic chemistry. In every case, they are characterized by a mechanism in which a hydrogen atom on one carbon atom exchanges places with a group X on an adjacent carbon: (formula; see text) Much experimental work indicates that an AdoCbl rearrangement is initiated by homolysis of the C-Co bond of the cofactor. The migrating hydrogen is then abstracted from the substrate by the resulting 5'-deoxyadenosyl radical, or by a second radical that is generated elsewhere at the active site, and, after the migration of group X, is returned to the product in a similar reaction. In at least some of the rearrangements, group X migration may occur via a cation radical intermediate that formed by the departure of X with its electrons, a process assisted by the unpaired electron left behind on the adjacent carbon after the abstraction of the migrating hydrogen. Once C-Co bond cleavage has initiated the reaction by producing a free radical at the active site, the corrin ring plays no further role in the rearrangements.     

Mechanistic studies of ubiquitin C-terminal hydrolase L1

Ubiquitin C-terminal hydrolases (UCHs) cleave Ub-X bonds (Ub is ubiquitin and X an alcohol, an amine, or a protein) through a thioester intermediate that is produced by nucleophilic attack of the Cys residue of a Cys-SH/His-Im catalytic diad. We are studying the mechanism of UCH-L1, a UCH that is implicated in Parkinson's disease, and now wish to report our initial findings. (i) Pre-steady-state kinetic studies for UCH-L1-catalyzed hydrolysis of Ub-AMC (AMC, 7-amido-4-methylcoumarin) indicate that k(cat) is rate-limited by acyl-enzyme formation. Thus, K(m) = K(s), the dissociation constant for the Michaelis complex, and k(cat) = k(2), the rate constant for acyl-enzyme formation. (ii) For K(assoc) (=K(s)(-)(1)), DeltaC(p) = -0.8 kcal mol(-)(1) deg(-)(1) and is consistent with coupling between substrate association and a conformational change of the enzyme. For k(2), DeltaS(++) = 0 and suggests that in the E-S, substrate and active site residues are precisely aligned for reaction. (iii) Solvent isotope effects are (D)K(assoc) = 0.5 and (D)k(2) = 0.9, suggesting that the substrate binds to a form of free enzyme in which the active site Cys exists as the thiol. In the resultant Michaelis complex, the diad has tautomerized to ion pair Cys-S(-)/His-ImH(+). Subsequent attack of thiolate produces the acyl-enzyme species. In contrast, isotope effects for association of UCH-L1 with transition-state analogue ubiquitin aldehyde suggest that an alternative mechanistic pathway can sometimes be available to UCH-L1 involving general base-catalyzed attack of Cys-SH by His-Im.     

Evaluation of the catalytic mechanism of the p21-activated protein kinase PAK2

The p21-activated kinases (PAKs) play important roles in diverse cellular processes. In the present study, we provide an in-depth kinetic analysis of one of the PAK family members, PAK2, in phosphorylation of a protein substrate, myelin basic protein (MBP), and a synthetic peptide substrate derived from LIM kinase, LIMKtide. Steady-state kinetic analysis of the initial reaction velocity of PAK2 phosphorylation of MBP is consistent with both randomly and compulsorily ordered mechanisms. Further kinetic studies carried out in various concentrations of sucrose revealed that solvent viscosities had no effect on k(cat)/K(m) for either ATP or MBP while k(cat) was highly sensitive to solvent viscosity, indicating that the enzymatic phosphorylation by PAK2 can be best interpreted by a rapid-equilibrium random bi-bi reaction model, and k(cat) is partially limited by both phosphoryl group transfer (31 s(-)(1)) and the product release (19 s(-)(1)). In the phosphorylation of LIMKtide, both k(cat) and k(cat)/K(m) were insensitive to solvent viscosity, and the product release (86 s(-)(1)) was much faster than the phosphoryl group transfer step (19 s(-)(1)). These studies suggest that the release of phospho-MBP product is likely partially rate determining for the PAK2-catalyzed reaction since the dissociation rate of products from the PAK2 active site for LIMKtide phosphorylation differs from that of MBP significantly. Such a mechanism is in contrast to the previously established kinetics for the phosphorylation of peptide substrates by cAMP-dependent kinase, in which this process is limited by the release of ADP but not the phospho-peptide product. These results complement previous structure-function studies of PAKs and provide important insight for mechanistic interpretation of the kinase functions.     

Tyr115, gln165 and trp209 contribute to the 1, 2-epoxy-3-(p-nitrophenoxy)propane-conjugating activity of glutathione S-transferase cGSTM1-1

We investigated the epoxidase activity of a class mu glutathione S-transferase (cGSTM1-1), using 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) as substrate. Trp209 on the C-terminal tail, Arg107 on the alpha4 helix, Asp161 and Gln165 on the alpha6 helix of cGSTM1-1 were selected for mutagenesis and kinetic studies. A hydrophobic side-chain at residue 209 is needed for the epoxidase activity of cGSTM1-1. Replacing Trp209 with histidine, isoleucine or proline resulted in a fivefold to 28-fold decrease in the k(cat)(app) of the enzyme, while a modest 25 % decrease in the k(cat)(app) was observed for the W209F mutant. The rGSTM1-1 enzyme has serine at the correponding position. The k(cat)(app) of the S209W mutant is 2. 5-fold higher than that of the wild-type rGSTM1-1. A charged residue is needed at position 107 of cGSTM1-1. The K(m)(app)(GSH) of the R107L mutant is 38-fold lower than that of the wild-type enzyme. On the contrary, the R107E mutant has a K(m)(app)(GSH) and a k(cat)(app) that are 11-fold and 35 % lower than those of the wild-type cGSTM1-1. The substitutions of Gln165 with Glu or Leu have minimal effect on the affinity of the mutants towards GSH or EPNP. However, a discernible reduction in k(cat)(app) was observed. Asp161 is involved in maintaining the structural integrity of the enzyme. The K(m)(app)(GSH) of the D161L mutant is 616-fold higher than that of the wild-type enzyme. In the hydrogen/deuterium exchange experiments, this mutant has the highest level of deuteration among all the proteins tested.We also elucidated the structure of cGSTM1-1 co-crystallized with the glutathionyl-conjugated 1, 2-epoxy-3-(p-nitrophenoxy)propane (EPNP) at 2.8 A resolution. The product found in the active site was 1-hydroxy-2-(S-glutathionyl)-3-(p-nitrophenoxy)propane, instead of the conventional 2-hydroxy isomer. The EPNP moiety orients towards Arg107 and Gln165 in dimer AB, and protrudes into a hydrophobic region formed by the loop connecting beta1 and alpha1 and part of the C-terminal tail in dimer CD. The phenoxyl ring forms strong ring stacking with the Trp209 side-chain in dimer CD. We hypothesize that these two conformations represent the EPNP moiety close to the initial and final stages of the reaction mechanism, respectively.     

Kinetics of cytochrome P450 2E1-catalyzed oxidation of ethanol to acetic acid via acetaldehyde.

The P450 2E1-catalyzed oxidation of ethanol to acetaldehyde is characterized by a kinetic deuterium isotope effect that increases K(m) with no effect on k(cat), and rate-limiting product release has been proposed to account for the lack of an isotope effect on k(cat) (Bell, L. C., and Guengerich, F. P. (1997) J. Biol. Chem. 272, 29643-29651). Acetaldehyde is also a substrate for P450 2E1 oxidation to acetic acid, and k(cat)/K(m) for this reaction is at least 1 order of magnitude greater than that for ethanol oxidation to acetaldehyde. Acetic acid accounts for 90% of the products generated from ethanol in a 10-min reaction, and the contribution of this second oxidation has been overlooked in many previous studies. The noncompetitive intermolecular kinetic hydrogen isotope effects on acetaldehyde oxidation to acetic acid ((H)(k(cat)/K(m))/(D)(k(cat)/K(m)) = 4.5, and (D)k(cat) = 1.5) are comparable with the isotope effects typically observed for ethanol oxidation to acetaldehyde, and k(cat) is similar for both reactions, suggesting a possible common catalytic mechanism. Rapid quench kinetic experiments indicate that acetic acid is formed rapidly from added acetaldehyde (approximately 450 min(-1)) with burst kinetics. Pulse-chase experiments reveal that, at a subsaturating concentration of ethanol, approximately 90% of the acetaldehyde intermediate is directly converted to acetic acid without dissociation from the enzyme active site. Competition experiments suggest that P450 2E1 binds acetic acid and acetaldehyde with relatively high K(d) values, which preclude simple tight binding as an explanation for rate-limiting product release. The existence of a rate-determining step between product formation and release is postulated. Also proposed is a conformational change in P450 2E1 occurring during the course of oxidation and the discrimination of P450 2E1 between acetaldehyde and its hydrated form, the gem-diol. This multistep P450 reaction is characterized by kinetic control of individual reaction steps and by loose binding of all ligands.     

When a spectator turns killer: suicidal electron transfer from cobalamin in methylmalonyl-CoA mutase

Methylmalonyl-CoA mutase belongs to the class of adenosylcobalamin (AdoCbl)-dependent carbon skeleton isomerases and catalyzes the rearrangement of methylmalonyl-CoA to succinyl-CoA. In this study, we have evaluated the contribution of the active site residue, R207, in the methylmalonyl-CoA mutase-catalyzed reaction. The R207Q mutation results in a 10(4)-fold decrease in k(cat) and >30-fold increase in the K(M) for the substrate, methylmalonyl-CoA. R207 and the active site residue, Y89, are within hydrogen bonding distance to the carboxylate of the substrate. In the closely related isomerase, isobutyryl-CoA mutase the homologous residues are F80 and Q198, respectively. We therefore characterized the ability of the double mutant (Y89F/R207Q) of methylmalonyl-CoA mutase as well as of the single mutants (Y89F and R207Q) to catalyze the rearrangement of n-butyryl-CoA to isobutyryl-CoA. While none of the mutant enzymes is capable of isomerizing these substrates, the R207Q (single and double) mutants exhibited irreversible inactivation upon incubation with either n-butyryl-CoA or isobutyryl-CoA. The two products observed during inactivation under both aerobic and strictly anaerobic conditions were 5'-deoxyadenosine and hydroxocobalamin, which suggested internal electron transfer from cob(II)alamin to the substrate or the 5'-deoxyadenosyl radical. Deuterium transfer from substrate to deoxyadenosine demonstrated that the substrate radical is formed and is presumably the acceptor in the electron-transfer reaction from cob(II)alamin. These studies provide evidence for the critical role of active site residues in controlling radical reactivity and thereby suppressing inactivating side reactions.     

A Birch-like mechanism in enzymatic benzoyl-CoA reduction: a kinetic study of substrate analogues combined with an ab initio model

Benzoyl-CoA reductase from the anaerobic bacterium Thauera aromatica catalyzes the ATP-driven two-electron reduction of the aromatic moiety of benzoyl-CoA. A Birch mechanism involving alternate one-electron and one-proton transfer steps to the aromatic ring was previously proposed for benzoyl-CoA reductase. Due to the high redox barrier, the first electron transfer step yielding a radical anion is considered the rate-limiting step in this reaction. Focusing on the mechanism of substrate reduction, this work combines the kinetic analysis of a number of substrate analogues with a model based on the ab initio calculated electron density of the radical anion of benzoyl-CoA, a transition state model of the proposed Birch mechanism. Both K(m) and k(cat) of ortho-substituted benzoyl-CoA increased in parallel with the substituent's acceptor strength (F > Cl = H > OH > NH(2)). Among the isomers of monofluorobenzoyl-CoA, reduction rates decreased in the following order: ortho > meta > para; the K(m) values increased in the following order: meta > ortho > para. Five-ring heteroaromatic acid thiol esters were reduced in the following order: thiophene > furan > pyrrole; the 2-isomers are reduced much faster than the 3-isomers. Most of these results could be rationalized by the model. A Hammett plot indicated that the reaction mechanism is only slightly polar, suggesting the involvement of a partial protonation of the carbonyl oxygen of benzoyl-CoA and/or a simultaneous transfer of the first electron and proton. Surprisingly, benzoyl-CoA reductase exhibited a hydrogen kinetic isotope effect on k(cat) for pyridine-2-carbonyl-CoA (2.1) but only a negligible one for benzoyl-CoA (1.2), indicating that pyridine-2-carbonyl-CoA reduction proceeds according to a varied mechanism.     

Insight into the catalysis of hydrolysis of four newly synthesized substrates by papain: a proton inventory study

We synthesized the following four new peptide substrates, Suc-Phe-Leu-pNA, Suc-Phe-Leu-NMec, Suc-Phe-Leu-ONPh, and Pht-Phe-Leu-pNA, and we applied the proton inventory method to their hydrolysis by papain. Useful relationships between the rate constants of the catalytic reaction have been established and contributed to the elucidation of the hydrolytic mechanism of papain. For all amide substrates, the parameter K(S) and the rate constants k(1), k(-)(1), and k(2) were estimated. Moreover, it was found that k(cat)/K(m) = k(1) for all four substrates, while two exchangeable hydrogenic sites, one in the ground state and another in the transition state, generate an inverse isotope effect during the reaction governed by this parameter. The proton inventories of both k(2) and k(3) are essentially linear, whatever the acyl moiety and/or the leaving group of the substrate. The proton inventories of K(S) are also essentially linear for all amide substrates, while the observed large isotope effect of about 3 to 9 originates from a single hydrogenic site in the product state. This latter, in agreement to both the small transition state fractionation factors found for k(cat)/K(m) (or k(1)) and the unit ground-state fractionation factors found for k(2), argues for the formation of a tetrahedral adduct during the reaction governed by the k(1) parameter. Furthermore, papain acts as a one-proton catalyst during acylation or deacylation, both of which proceed through similar concerted reaction pathways, where a nucleophilic attack is accompanied by the movement of one proton.     

Catalytic reaction pathway for the mitogen-activated protein kinase ERK2

The structural, functional, and regulatory properties of the mitogen-activated protein kinases (MAP kinases) have long attracted considerable attention owing to the critical role that these enzymes play in signal transduction. While several MAP kinase X-ray crystal structures currently exist, there is by comparison little mechanistic information available to correlate the structural data with the known biochemical properties of these molecules. We have employed steady-state kinetic and solvent viscosometric techniques to characterize the catalytic reaction pathway of the MAP kinase ERK2 with respect to the phosphorylation of a protein substrate, myelin basic protein (MBP), and a synthetic peptide substrate, ERKtide. A minor viscosity effect on k(cat) with respect to the phosphorylation of MBP was observed (k(cat) = 10 +/- 2 s(-1), k(cat)(eta) = 0.18 +/- 0.05), indicating that substrate processing occurs via slow phosphoryl group transfer (12 +/- 4 s(-1)) followed by the faster release of products (56 +/- 4 s(-1)). At an MBP concentration extrapolated to infinity, no significant viscosity effect on k(cat)/K(m(ATP)) was observed (k(cat)/K(m(ATP)) = 0.2 +/- 0.1 microM(-1) s(-1), k(cat)/K(m(ATP))(eta) = -0.08 +/- 0.04), consistent with rapid-equilibrium binding of the nucleotide. In contrast, at saturating ATP, a full viscosity effect on k(cat)/K(m) for MBP was apparent (k(cat)/K(m(MBP)) = 2.4 +/- 1 microM(-1) s(-1), k(cat)/K(m(MBP))(eta) = 1.0 +/- 0.1), while no viscosity effect was observed on k(cat)/K(m) for the phosphorylation of ERKtide (k(cat)/K(m(ERKtide)) = (4 +/- 2) x 10(-3) microM(-1) s(-1), k(cat)/K(m(ERKtide))(eta) = -0.02 +/- 0.02). This is consistent with the diffusion-limited binding of MBP, in contrast to the rapid-equilibrium binding of ERKtide, to form the ternary Michaelis complex. Calculated values for binding constants show that the estimated value for K(d(MBP)) (/= 1.5 mM). The dramatically higher catalytic efficiency of MBP in comparison to that of ERKtide ( approximately 600-fold difference) is largely attributable to the slow dissociation rate of MBP (/=56 s(-1)), from the ERK2 active site.     

The mechanism of DNA cytosine-5 methylation. Kinetic and mutational dissection of Hhai methyltransferase

Kinetic and binding studies involving a model DNA cytosine-5-methyltransferase, M.HhaI, and a 37-mer DNA duplex containing a single hemimethylated target site were applied to characterize intermediates on the reaction pathway. Stopped-flow fluorescence studies reveal that cofactor S-adenosyl-l-methionine (AdoMet) and product S-adenosyl-l-homocysteine (AdoHcy) form similar rapidly reversible binary complexes with the enzyme in solution. The M.HhaI.AdoMet complex (k(off) = 22 s(-)1, K(D) = 6 microm) is partially converted into products during isotope-partitioning experiments, suggesting that it is catalytically competent. Chemical formation of the product M.HhaI.(Me)DNA.AdoHcy (k(chem) = 0.26 s(-)1) is followed by a slower decay step (k(off) = 0.045 s(-)1), which is the rate-limiting step in the catalytic cycle (k(cat) = 0.04 s(-)1). Analysis of reaction products shows that the hemimethylated substrate undergoes complete (>95%) conversion into fully methylated product during the initial burst phase, indicating that M.HhaI exerts high binding selectivity toward the target strand. The T250N, T250D, and T250H mutations, which introduce moderate perturbation in the catalytic site, lead to substantially increased K(D)(DNA(ternary)), k(off)(DNA(ternary)), K(M)(AdoMet(ternary)) values but small changes in K(D)(DNA(binary)), K(D)(AdoMet(binary)), k(chem), and k(cat). When the target cytosine is replaced with 5-fluorocytosine, the chemistry step leading to an irreversible covalent M.HhaI.DNA complex is inhibited 400-fold (k(chem)(5FC) = 0.7 x 10(-)3 s(-)1), and the Thr-250 mutations confer further dramatic decrease of the rate of the covalent methylation k(chem). We suggest that activation of the pyrimidine ring via covalent addition at C-6 is a major contributor to the rate of the chemistry step (k(chem)) in the case of cytosine but not 5-fluorocytosine. In contrast to previous reports, our results imply a random substrate binding order mechanism for M.HhaI.     

A novel reaction between adenosylcobalamin and 2-methyleneglutarate catalyzed by glutamate mutase

We describe a novel reaction of adenosylcobalamin that occurs when adenosylcobalamin-dependent glutamate mutase is reacted with the substrate analogue 2-methyleneglutarate. Although 2-methyleneglutarate is a substrate for the closely related adenosylcobalamin-dependent enzyme 2-methyleneglutarate mutase, it reacts with glutamate mutase to cause time-dependent inhibition of the enzyme. Binding of 2-methyleneglutarate to glutamate mutase initiates homolysis of adenosylcobalamin. However, instead of the adenosyl radical proceeding to abstract a hydrogen from the substrate, which is the next step in all adenosylcobalamin-dependent enzymes, the adenosyl radical undergoes addition to the exo-methylene group to generate a tertiary radical at C-2 of methyleneglutarate. This radical has been characterized by EPR spectroscopy with regiospecifically (13)C-labeled methyleneglutarates. Irreversible inhibition of the enzyme appears to be a complicated process, and the detailed chemical and kinetic mechanism remains to be elucidated. The kinetics of this process suggest that cob(II)alamin may reduce the enzyme-bound organic radical so that stable adducts between the adenosyl moiety of the coenzyme and 2-methyleneglutarate are formed.     

Thermostability of native and pegylated Myceliophthora thermophila laccase in aqueous and mixed solvents

A commercial preparation of laccase (EC 1.10.3.2), cloned from Myceliophthora thermophila and expressed in Aspergillus oryzae (MtL), was purified and modified by conjugation with poly(ethylene glycol) (M(r) = 5000) and is labeled PEG-MtL. Native enzyme was found to have a molecular mass of 80 kDa, as determined by gel filtration, and 110 kDa, by SDS-PAGE. The oxidative dimerization of 2,6-dimethoxyphenol (DMP) to produce the corresponding dibenzoquinone was catalyzed by MtL in a manner comparable to that for a diffusion-controlled reaction (k(cat)/K(M) approximately = 10(8) M(-)(1) s(-)(1) and E(a) approximately = 18 kJ M(-)(1)). PEG-MtL was found, by TNBS titration, to have blocked 54% of lysine groups; its hydrodynamic and charge properties were different from those of MtL. Catalytic efficiency (k(cat)/K(M)) of PEG-MtL was similar to that of MtL with DMP as substrate; however, k(cat)/K(M) was 2-fold reduced for the reaction in which 2',2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is oxidized to form a radical cation. E(a) values were similar in both enzyme preparations when assayed in buffered solutions. Far-UV CD spectra were similar for MtL and PEG-MtL and consistent with a protein rich in beta-sheet structure with negligible content of alpha-helices. A blue shift of near-UV CD spectrum for PEG-MtL as compared to MtL was consistent with the decreased polarity of the tyrosyl side chains upon PEG conjugation. Also the blue band of the copper active site was shifted from lambda approximately 610 nm (MtL) to lambda approximately 575 nm (PEG-MtL). Scanning microcalorimetry showed small denaturation enthalpies (6.3 and 7.5 J g(-)(1) for MtL and PEG-MtL, respectively), indicating the high stability of the beta-sheet folding pattern of laccases. However, PEG-MtL proved to be more stable, its half-denaturation temperature being 2 degrees C higher than that of MtL. In 30% alcohol, pegylated laccase showed slower enzyme-activity decay rates than the unmodified enzyme; this behavior was caused by a decrease in the activation entropy of the denaturation reaction. Results can be explained by entropic stabilization by PEG conjugation because of the restricted motion of some surface amino acid side chains, which results in a more stable active site.     

Temperature effects on the catalytic efficiency, rate enhancement, and transition state affinity of cytidine deaminase, and the thermodynamic consequences for catalysis of removing a substrate "anchor"

To obtain a clearer understanding of the forces involved in transition state stabilization by Escherichia coli cytidine deaminase, we investigated the thermodynamic changes that accompany substrate binding in the ground state and transition state for substrate hydrolysis. Viscosity studies indicate that the action of cytidine deaminase is not diffusion-limited. Thus, K(m) appears to be a true dissociation constant, and k(cat) describes the chemical reaction of the ES complex, not product release. Enzyme-substrate association is accompanied by a loss of entropy and a somewhat greater release of enthalpy. As the ES complex proceeds to the transition state (ES), there is little further change in entropy, but heat is taken up that almost matches the heat that was released with ES formation. As a result, k(cat)/K(m) (describing the overall conversion of the free substrate to ES is almost invariant with changing temperature. The free energy barrier for the enzyme-catalyzed reaction (k(cat)/K(m)) is much lower than that for the spontaneous reaction (k(non)) (DeltaDeltaG = -21.8 kcal/mol at 25 degrees C). This difference, which also describes the virtual binding affinity of the enzyme for the activated substrate in the transition state (S), is almost entirely enthalpic in origin (DeltaDeltaH = -20.2 kcal/mol), compatible with the formation of hydrogen bonds that stabilize the ES complex. Thus, the transition state affinity of cytidine deaminase increases rapidly with decreasing temperature. When a hydrogen bond between Glu-91 and the 3'-hydroxyl moiety of cytidine is disrupted by truncation of either group, k(cat)/K(m) and transition state affinity are each reduced by a factor of 10(4). This effect of mutation is entirely enthalpic in origin (DeltaDeltaH approximately 7.9 kcal/mol), somewhat offset by a favorable change in the entropy of transition state binding. This increase in entropy is attributed to a loss of constraints on the relative motions of the activated substrate within the ES complex. In an Appendix, some objections to the conventional scheme for transition state binding are discussed.     

Isotope effects for deuterium transfer between substrate and coenzyme in adenosylcobalamin-dependent glutamate mutase

A key step in the mechanism of all adenosylcobalamin-dependent enzymes is the abstraction of a hydrogen atom from the substrate by a 5'-deoxyadenosyl radical generated by homolytic fission of the coenzyme cobalt-carbon bond. We have investigated the isotope effects associated with this process for glutamate mutase reacting with deuterated glutamate. The kinetics of deuterium incorporation into 5'-deoxyadenosine (5'-dA) during the reaction were followed by rapid chemical quench, using HPLC and electrospray mass spectrometry to analyze the 5'-dA formed. The kinetics of 5'-dA formation are biphasic, comprising a rapid phase k(app) = 37 +/- 3 s(-)(1) and a slower phase k(app) = 0.9 +/- 0.4 s(-)(1). The mass spectral data clearly show that the faster phase is associated with the formation of monodeuterated 5'-dA whereas the slower phase is associated with the incorporation of a second and then a third deuterium into 5'-dA. This observation implies that a large inverse equilibrium secondary isotope effect is associated with the formation of 5'-dA from adenosylcobalamin. The primary deuterium kinetic isotope effects on V and V/K for the formation of 5'-dA were determined from time-based and competition experiments. (D)V = 2.4 +/-0.4 whereas (D)(V/K) = 10 +/- 0.4, implying that an isotopically insensitive step is partially rate-determining. The additional data provided by these experiments cause us to revise our interpretation of earlier UV-visible stopped-flow kinetic measurements of AdoCbl homolysis obtained with deuterated substrates.