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PubMed1985

Symmetry and asymmetry in mandelate racemase catalysis.

Kinetic properties of mandelate racemase catalysis (Vmax, Km, deuterium isotope effects, and pH profiles) were all measured in both directions by the circular dichroic assay of Sharp et al. [Sharp, T. R., Hegeman, G. D., & Kenyon, G. L. (1979) Anal. Biochem. 94, 329]. These results, along with those of studying interactions of mandelate racemase with resolved, enantiomeric competitive inhibitors [(R)- and (S)-alpha-phenylglycerates], indicate a high degree of symmetry in both binding and catalysis. Racemization of either enantiomer of mandelate in D2O did not show an overshoot region of molecular ellipticity in circular dichroic measurements upon approach to equilibrium. Both the absence of such an overshoot region and the high degree of kinetic symmetry are consistent with a one-base acceptor mechanism for mandelate racemase. On the other hand, results of irreversible inhibition with partially resolved, enantiomeric affinity labels [(R)- and (S)-alpha-phenylglycidates] reveal a "functional asymmetry" at the active site. Mechanistic proposals, consistent with these results, are presented.

Circular Dichroism↗
PubMed1988

Kinetics and mechanism of benzoylformate decarboxylase using 13C and solvent deuterium isotope effects on benzoylformate and benzoylformate analogues.

Benzoylformate decarboxylase (benzoylformate carboxy-lyase, BFD; EC 4.1.1.7) from Pseudomonas putida is a thiamine pyrophosphate (TPP) dependent enzyme which converts benzoylformate to benzaldehyde and carbon dioxide. The kinetics and mechanism of the benzoylformate decarboxylase reaction were studied by solvent deuterium and 13C kinetic isotope effects with benzoylformate and a series of substituted benzoylformates (pCH3O, pCH3, pCl, and mF). The reaction was found to have two partially rate-determining steps: initial tetrahedral adduct formation (D2O sensitive) and decarboxylation (13C sensitive). Solvent deuterium and 13C isotope effects indicate that electron-withdrawing substituents (pCl and mF) reduce the rate dependence upon decarboxylation such that decreased 13(V/K) effects are observed. Conversely, electron-donating substituents increase the rate dependence upon decarboxylation such that a larger 13(V/K) is seen while the D2O effects on V and V/K are not dramatically different from those for benzoylformate. All of the data are consistent with substituent stabilization or destabilization of the carbanionic intermediate (or carbanion-like transition state) formed during decarboxylation. Additional information regarding the mechanism of the enzymic reaction was obtained from pH studies on the reaction of benzoylformate and the binding of competitive inhibitors. These studies suggest that two enzymic bases are required to be in the correct protonation state (one protonated and one unprotonated) for optimal binding of substrate (or inhibitors).

Benzoates↗
PubMed1989

Selection and characterization of a mutant of the cloned gene for mandelate racemase that confers resistance to an affinity label by greatly enhanced production of enzyme.

The plasmid pSCR1 containing the gene for mandelate racemase (EC 5.1.2.2) from Pseudomonas putida (ATCC 12633) allows Pseudomonas aeruginosa (ATCC 15692) to grow on (R)-mandelate as its sole carbon source [Ransom, S. C., Gerlt, J. A., Powers, V. M., & Kenyon, G. L. (1988) Biochemistry 27, 540]; the chromosome of the P. aeruginosa host apparently does not contain the gene for mandelate racemase but does contain genes for the remaining enzymes in the mandelate pathway and enables growth on (S)-mandelate as carbon source. However, in the presence of alpha-phenylglycidate, an active-site-directed irreversible inhibitor (affinity label) of mandelate racemase, P. aeruginosa transformed with pSCR1 can utilize (S)-mandelate but not (R)-mandelate as carbon source. This inhibition of growth on (R)-mandelate provides a metabolic selection for mutants that are resistant to alpha-phenylglycidate. When (R)-mandelate is used as carbon source and alpha-phenylglycidate is present, a few colonies of P. aeruginosa transformed with pSCR1 grow slowly and appear on plates after several days. The plasmid isolated from these cells confers resistance to alpha-phenylglycidate on newly transformed cells of P. aeruginosa. This resistance to the affinity label is not due to a mutation within the primary structure of the enzyme. A single base change (C----A) located 87 bp upstream of the initiation codon for the gene for mandelate racemase was detected in three independent isolates of alpha-phenylglycidate-resistant colonies and appears responsible for a 30-fold increase in the amount of mandelate racemase encoded by the gene contained in the plasmid.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1990

Oxidation of monomethoxylated aromatic compounds by lignin peroxidase: role of veratryl alcohol in lignin biodegradation.

Lignin peroxidase (LiP), an extracellular heme enzyme from the lignin-degrading fungus Phanerochaete chrysosporium, catalyzes the H2O2-dependent oxidation of a variety of nonphenolic lignin model compounds. The oxidation of monomethoxylated lignin model compounds, such as anisyl alcohol (AA), and the role of veratryl alcohol (VA) in LiP reactions were studied. AA oxidation reached a maximum at relatively low H2O2 concentrations, beyond which the extent of the reactions decreased. The presence of VA did not affect AA oxidation at low molar ratios of H2O2 to enzyme; however, at ratios above 100, the presence of VA abolished the decrease in AA oxidation. Addition of stoichiometric amounts of AA to LiP compound II (LiPII) resulted in its reduction to the native enzyme at rates that were significantly faster than the spontaneous rate of reduction, indicating that AA and other monomethoxylated aromatics are directly oxidized by LiP, albeit slowly. Under steady-state conditions in the presence of excess H2O2 and VA, a visible spectrum for LiPII was obtained. In contrast, under steady-state conditions in the presence of AA a visible spectrum was obtained for LiPIII*, a noncovalent complex of LiPIII and H2O2. AA competitively inhibited the oxidation of VA by LiP; the Ki for AA inhibition was 32 microM. Addition of VA to LiPIII* resulted in its conversion to the native enzyme. In contrast, AA did not convert LiPIII* to the native enzyme; instead, LiPIII* was bleached in the presence of AA. Thus, AA does not protect LiP from inactivation by H2O2.(ABSTRACT TRUNCATED AT 250 WORDS)

Anisoles↗
PubMed1982

Involvement of arginine residues in the allosteric activation of Escherichia coli ADP-glucose synthetase.

Inactivation of Escherichia coli ADP-glucose synthetase (EC 2.7.2.27) by the arginine-specific reagents cyclohexanedione and phenylglyoxal resulted primarily from interference with normal allosteric activation. Partial modification by phenylglyoxal resulted in a lessened ability of fructose 1,6-bisphosphate (fructose-P2) to stimulate and of 5'-AMP (5'-adenylate) to inhibit enzymic activity. The apparent affinity for fructose-P2 and the Vmax at saturating fructose-P2 concentrations were decreased by the arginine modification. Fructose-P2, 5'-adenylate, and several other allosteric effectors were able to partially protect the enzyme from inactivation. However, catalytic activity was not decreased by arginine modification under conditions where the enzyme was assayed in the absence of fructose-P2. The two arginine-modifying reagents differed markedly in their reactivity with the enzyme. Cyclohexanedione inactivated the enzyme quite slowly and eventually reacted with at least 14 of the 32 arginines present per subunit. Phenylglyoxal was some 50-fold more effective in inactivation, but it modified only one arginine residue per subunit.

Adenosine Monophosphate↗
PubMed1977

Mandelate racemase from Pseudomonas putida. Absence of detectable intermolecular proton transfer accompanying racemization.

An equimolar mixture of DL-[alpha-2H]- and DL-[alpha-13C]mandelate, when incubated with mandelate racemase (EC 5.1.2.2), shows conversion of singly labeled mandelate to unlabeled mandelate, due to solvent exchange of the alpha proton, while the level of doubly labeled mandelate remains at a constant low level. Similarly, an equimolar mixture of unlabeled and DL-[alpha-2H,alpha-13C]mandelate, when incubated with the enzyme, shows conversion of doubly labeled mandelate to singly labeled mandelate, due to solvent exchange, while the level of unlabeled mandelate remains constant at 50%. Incubation of an equimolar mixture of DL-[alpha-3H]mandelate and DL-rho-chloromandelate. These results indicate that mandelate racemase does not catalyze an intermolecular proton transfer to achieve racemization. These data are necessary, but not sufficient, results to indicate that mandelate racemase operates via a one-acceptor mechanism, in which the proton abstracted from one stereochemical face of a substrate molecule is returned to the opposite face of the same carbon of the substrate molecule.

Carbon Isotopes↗
PubMed1975

Mandelate racemase from Pseudomonas putida. Magnetic resonance and kinetic studies of the mechanism of catalysis.

The interactions of mandelate racemase with divalent metal ion, substrate, and competitive inhibitors were investigated. The enzyme was found by electron paramagnetic resonance (EPR) to bind 0.9 Mn2+ ion per subunit with a dissociation constant of 8 muM, in agreement with its kinetically determined activator constant. Also, six additional Mn2+ ions were found to bind to the enzyme, much more weakly, with a dissociation constant of 1.5 mM. Binding to the enzyme at the tight site enhances the effect of Mn2+ on the longitudinal relaxation rate (1/T1p) of water protons by a factor of 11.9 at 24.3 MHz. From the frequency dependence of 1/T1p, it was determined that there are similar to 3 water ligands on enzyme-bound Mn2+ which exchange at a rate larger than or equal to 10-7 sec-1. The correlation time for enzyme-bound Mn2+-water interaction is frequency-dependent, indicating it to be dominated by the electron spin relaxation time of Mn2+. Formation of the ternary enzyme-Mn2+-mandelate complex decreases the number of fast exchanging water ligands by similar to 1, but does not affect tau-c, suggesting the displacement or occlusion of a water ligand. The competitive inhibitors D,L-alpha-phenylglycerate and salicylate produce little or no change in the enzyme-Mn2+-H2O interaction, but ternary complexes are detected indirectly by changes in the dissociation constant of the enzyme-Mn2+ complex and by mutual competition experiments. In all cases the dissociation constants of substrates and competitive inhibitors from ternary complexes determined by magnetic resonance titrations agree with K-M and K-i values determined kinetically and therefore reflect kinetically active complexes. From the paramagnetic effects of Mn2+ on 1/T1 and 1/T2 of the 13C-enriched carbons of 1-[13C]-D,L-mandelate and 2-[13C]-D,L-mandelate, Mn2+ to carboxylate carbon and Mn2+ to carbinol carbon distances of 2.93 plus or minus 0.04 and 2.71 plus or minus 0.04 A, respectively, were calculated, indicating bidentate chelation in the binary Mn2+-mandelate complex. In the active ternary complex of enzyme, Mn2+, and D,L-mandelate, these distances increase to 5.5 plus or minus 0.2 and 7.2 plus or minus 0.2 A, respectively, indicating the presence of at least 98.9% of a second sphere complex in which Mn2+, and C1 and C2 carbon atoms are in a linear array. The water relaxation data suggest that a water ligand is immobilized between the enzyme-bound Mn2+ and the carboxylate of the bound substrate. This intervening water ligand may polarize or protonate the carboxyl group. From 1/T2p the rate of dissociation of the substrate from this ternary complex (larger than or equal to 5.6 times 10-4 sec-1) is at least 52 times greater than the maximal turnover number of the enzyme (1070 sec-1), indicating that the complex detected by nuclear magnetic resonance (NMR) is kinetically competent to participate in catalysis. Relationships among the microscopic rate constants are considered.

Binding, Competitive↗
PubMed2002

Arginine 165/arginine 277 pair in (S)-mandelate dehydrogenase from Pseudomonas putida: role in catalysis and substrate binding.

(S)-Mandelate dehydrogenase from Pseudomonas putida belongs to a FMN-dependent enzyme family that oxidizes (S)-alpha-hydroxyacids. Active site structures of three homologous enzymes, including MDH, show the presence of two conserved arginine residues in close juxtaposition (Arg165 and Arg277 in MDH). Arg277 has an important catalytic role; it stabilizes both the ground and transition states through its positive charge as well as a hydrogen bond [Lehoux, I. E., and Mitra, B. (2000) Biochemistry 39, 10055-10065]. In this study, we examined the role of Arg165 and the overall importance of the Arg165/Arg277 pair. Single mutants at Arg165 as well as double mutants at Arg165 and Arg277 were characterized. Our results show that Arg165 has a role similar to, but less critical than, that of Arg277. It stabilizes the transition state through its positive charge and the ground state through a charge-independent interaction, most likely, a hydrogen bond. Though the k(cat)s for the charge-conserved mutants, R165K and R277K, were only 3-5-fold lower than those of wild-type MDH (wtMDH), the k(cat) for R165K/R277K was approximately 350-fold lower. Thus, at least one arginine residue is required for the optimal substrate orientation and catalysis. Stopped-flow studies show that the FMN reduction step is completely rate-limiting for both wtMDH and the arginine mutants, with the possible exception of R165E. Substrate isotope effects indicate that the carbon-hydrogen bond-breaking step is only partially rate-limiting for wtMDH but fully rate-limiting for the mutants. pH profiles of R165M conclusively show that the pK(a) of 9.3 in free wtMDH does not belong to Arg165.

Alcohol Oxidoreductases↗
PubMed2003

Structural and kinetic analysis of catalysis by a thiamin diphosphate-dependent enzyme, benzoylformate decarboxylase.

Benzoylformate decarboxylase is a member of the family of enzymes that are dependent on the cofactor thiamin diphosphate. A structure of this enzyme binding (R)-mandelate, a competitive inhibitor, suggests that at least two hydrogen bonds are formed between the substrate, benzoylformate, and active site side chains. The first is between the carboxylate group of benzoylformate and the hydroxyl group of S26, and the second is between carbonyl group of the substrate and an imidazole nitrogen of H70. Steady-state kinetic studies indicate that the catalytic parameters are strongly affected in three active site mutants, S26A, H70A, and H281A. The K(m) of S26A was increased most dramatically, 25-fold more than that of the wild-type enzyme, while the K(i) of (R)-mandelate was increased 100-fold, suggesting that the serine hydroxyl is important for substrate binding. The k(cat) of H70A is reduced more than 3 orders of magnitude, strongly implicating this residue in catalysis, and H281 showed significant, but smaller magnitude, effects on both K(m) and k(cat). Stopped-flow experiments using an alternative substrate, p-nitrobenzoylformate, lead to kinetic resolution of the fate of key thiamin diphosphate-bound intermediates. Together, the experimental results suggest the following roles for residues in the active site. The residue H70 is important for the protonation of the 2-alpha-mandelyl-ThDP intermediate, thereby assisting in decarboxylation, and for the deprotonation of the 2-alpha-hydroxybenzyl-ThDP intermediate, aiding product release. H281 is involved in protonation of the enamine. Surprisingly, S26 appears to be involved not only in substrate binding but also in other steps of the reaction.

Alanine↗
PubMed2005

Perturbing the hydrophobic pocket of mandelate racemase to probe phenyl motion during catalysis.

Mandelate racemase (MR, EC 5.1.2.2) from Pseudomonas putida catalyzes the Mg(2+)-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Crystal structures of MR reveal that the phenyl group of all ground-state ligands is located within a hydrophobic cavity, remote from the site of proton abstraction. MR forms numerous electrostatic and H-bonding interactions with the alpha-OH and carboxyl groups of the substrate, suggesting that these polar groups may remain relatively fixed in position during catalysis while the phenyl group is free to move between two binding sites [i.e., the R-pocket and the S-pocket for binding the phenyl group of (R)-mandelate and (S)-mandelate, respectively]. We show that MR binds benzilate (K(i) = 0.67 +/- 0.12 mM) and (S)-cyclohexylphenylglycolate (K(i) = 0.50 +/- 0.03 mM) as competitive inhibitors with affinities similar to that which the enzyme exhibits for the substrate. Therefore, the active site can simultaneously accommodate two phenyl groups, consistent with the existence of an R-pocket and an S-pocket. Wild-type MR exhibits a slightly higher affinity for (S)-mandelate [i.e., K(m)(S)(-)(man) < K(m)(R)(-)(man)] but catalyzes the turnover of (R)-mandelate slightly more rapidly (i.e., k(cat)(R)(-->)(S) > k(cat)(S)(-->)(R)). Upon introduction of steric bulk into the S-pocket using site-directed mutagenesis (i.e., the F52W, Y54W, and F52W/Y54W mutants), this catalytic preference is reversed. Although the catalytic efficiency (k(cat)/K(m)) of all the mutants was reduced (11-280-fold), all mutants exhibited a higher affinity for (R)-mandelate than for (S)-mandelate, and higher turnover numbers with (S)-mandelate as the substrate, relative to those with (R)-mandelate. (R)- and (S)-2-hydroxybutyrate are expected to be less sensitive to the additional steric bulk in the S-pocket. Unlike those for mandelate, the relative binding affinities for these substrate analogues are not reversed. These results are consistent with steric obstruction in the S-pocket and support the hypothesis that the phenyl group of the substrate may move between an R-pocket and an S-pocket during racemization. These conclusions were also supported by modeling of the binary complexes of the wild-type and F52W/Y54W enzymes with the substrate analogues (R)- and (S)-atrolactate, and of wild-type MR with bound benzilate using molecular dynamics simulations.

Binding Sites↗
PubMed2004

Fragmentation of the conjugate base of 2-(1-hydroxybenzyl)thiamin: does benzoylformate decarboxylase prevent orbital overlap to avoid it?

The base-catalyzed addition of thiamin to benzaldehyde produces 2-(1-hydroxybenzyl)thiamin (HBnT), but in neutral solution HBnT undergoes base-catalyzed irreversible fragmentation into pyrimidine and thiazole derivatives. The fragmentation (rather than elimination) occurs in proportion to the extent that N1' is protonated or alkylated. Generating the conjugate base of HBnT by decarboxylation surprisingly leads to fragmentation independent of the state of N1'. Therefore, a cationic state at N1' specifically promotes removal of the C2alpha proton rather than the fragmentation process itself. It is suggested that benzoylformate decarboxylase, which generates a similar intermediate, exerts stereoelectronic control of the conformation of the carbanion, blocking fragmentation and facilitating protonation.

Carboxy-Lyases↗
PubMed1991

Analogues of oxybutynin. Synthesis and antimuscarinic and bladder activity of some substituted 7-amino-1-hydroxy-5-heptyn-2-ones and related compounds.

Oxybutynin chloride [4-(diethylamino)-2-butynyl alpha-cyclohexyl-alpha-hydroxybenzeneacetate hydrochloride, Ditropan] is widely used for the relief of symptoms in neurogenic bladder. This is a result of its combined anticholinergic, antispasmodic, and local anesthetic activities. In a study directed toward development of agents possessing the beneficial properties of oxybutynin, but having a longer duration of action, a series of metabolically more stable keto analogues of the parent ester, i.e. substituted 7-amino-1-hydroxy-5-heptyn-2-ones along with some analogues and derivatives, was prepared and evaluated for in vitro and in vivo antimuscarinic action in guinea pig preparations. Several members of the series were potent antimuscarinics having a longer duration of activity than that of oxybutynin in a guinea pig cystometrogram model. On the basis of its in vitro and in vivo antimuscarinic activity, coupled with a 5-fold greater duration of action than that of oxybutynin, 1-cyclobutyl-7-(dimethylamino)-1-hydroxy-1-phenyl-5-heptyn-2-one (14b) was selected for clinical evaluation.

Alkynes↗