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Substrate analogues as probes of the catalytic mechanism of L-mandelate dehydrogenase from Rhodotorula graminis.

ABSTRACT: A detailed kinetic analysis of the oxidation of mono-substituted mandelates catalysed by L-(+)-mandelate dehydrogenase (L-MDH) from Rhodotorula graminis has been carried out to elucidate the role of the substrate in the catalytic mechanism. Values of Km and kcat. (25 degrees C, pH 7.5) were determined for mandelate and eight substrate analogues. Values of the activation parameters, delta H++ and delta S++ (determined over the range 5-37 degrees C), for mandelate and all substrate analogues were compensatory resulting in similar low values for free energies of activation delta G++ (approx. 60 kJ.mol-1 at 298.15 K) in all cases. A kinetic-isotope-effect value of 1.1 +/- 0.1 was observed using D,L-[2-2H]mandelate as substrate and was invariant over the temperature range studied. The logarithm of kcat. values for the enzymic oxidation of mandelate and all substrate analogues (except 4-hydroxymandelate) showed good correlation with Taft's dual substituent constant omega (where omega = omega I + 0.64 omega +R) and gave a positive reaction constant value, rho, of 0.36 +/- 0.07. This linear free-energy relationship was verified by analysing the data using isokinetic methods. These findings support the hypothesis that the enzyme-catalysed reaction proceeds via the same transition state for each substrate and indicates that this transition state is relatively nonpolar but has an electron-rich centre at the alpha-carbon position.

SUBMITTER: Smekal O

PROVIDER: S-EPMC1137881 | biostudies-other | 1994 Feb

REPOSITORIES: biostudies-other

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Project description:D(--)-Mandelate dehydrogenase, the first enzyme of the mandelate pathway in the yeast Rhodotorula graminis, catalyses the NAD(+)-dependent oxidation of D(--)-mandelate to phenylglyoxylate. D(--)-2-(Bromoethanoyloxy)-2-phenylethanoic acid ['D(--)-bromoacetylmandelic acid'], an analogue of the natural substrate, was synthesized as a probe for reactive and accessible nucleophilic groups within the active site of the enzyme. D(--)-Mandelate dehydrogenase was inactivated by D(--)-bromoacetylmandelate in a psuedo-first-order process. D(--)-Mandelate protected against inactivation, suggesting that the residue that reacts with the inhibitor is located at or near the active site. Complete inactivation of the enzyme resulted in the incorporation of approx. 1 mol of label/mol of enzyme subunit. D(--)-Mandelate dehydrogenase that had been inactivated with 14C-labelled D(--)-bromoacetylmandelate was digested with trypsin; there was substantial incorporation of 14C into two tryptic-digest peptides, and this was lowered in the presence of substrate. One of the tryptic peptides had the sequence Val-Xaa-Leu-Glu-Ile-Gly-Lys, with the residue at the second position being the site of radiolabel incorporation. The complete sequence of the second peptide was not determined, but it was probably an N-terminally extended version of the first peptide. High-voltage electrophoresis of the products of hydrolysis of modified protein showed that the major peak of radioactivity co-migrated with N tau-carboxymethylhistidine, indicating that a histidine residue at the active site of the enzyme is the most likely nucleophile with which D(--)-bromoacetylmandelate reacts. D(--)-Mandelate dehydrogenase was incubated with phenylglyoxylate and either (4S)-[4-3H]NADH or (4R)-[4-3H]NADH and then the resulting D(--)-mandelate and NAD+ were isolated. The enzyme transferred the pro-R-hydrogen atom from NADH during the reduction of phenylglyoxylate. The results are discussed with particular reference to the possibility that this enzyme evolved by the recruitment of a 2-hydroxy acid dehydrogenase from another metabolic pathway.

Project description:The l-mandelate dehydrogenase (L-MDH) from the yeast Rhodotorula graminis is a mitochondrial flavocytochrome b2 which catalyses the oxidation of mandelate to phenylglyoxylate coupled with the reduction of cytochrome c. We have used the N-terminal sequence of the enzyme to isolate the gene encoding this enzyme using the PCR. Comparison of the genomic sequence with the sequence of cDNA prepared by reverse transcription PCR revealed the presence of 11 introns in the coding region. The predicted amino acid sequence indicates a close relationship with the flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala, with about 40% identity to each. The sequence shows that a key residue for substrate specificity in S. cerevisiae flavocytochrome b2, Leu-230, is replaced by Gly in L-MDH. This substitution is likely to play an important part in determining the different substrate specificities of the two enzymes. We have developed an expression system and purification protocol for recombinant L-MDH. In addition, we have expressed and purified the flavin-containing domain of L-MDH independently of its cytochrome domain. Detailed steady-state and pre-steady-state kinetic investigations of both L-MDH and its independently expressed flavin domain have been carried out. These indicate that L-MDH is efficient with both physiological (cytochrome c, kcat=225 s-1 at 25 degrees C) and artificial (ferricyanide, kcat=550 s-1 at 25 degrees C) electron acceptors. Kinetic isotope effects with [2-2H]mandelate indicate that H-C-2 bond cleavage contributes somewhat to rate-limitation. However, the value of the isotope effect erodes significantly as the catalytic cycle proceeds. Reduction potentials at 25 degrees C were measured as -120 mV for the 2-electron reduction of the flavin and -10 mV for the 1-electron reduction of the haem. The general trends seen in the kinetic studies show marked similarities to those observed previously with the flavocytochrome b2 (L-lactate dehydrogenase) from S. cerevisiae.

Project description:Mandelate racemase (MR, EC 5.1.2.2) from Pseudomonas putida catalyzes the Mg(2+)-dependent interconversion of the enantiomers of mandelate, stabilizing the altered substrate in the transition state by 26 kcal/mol relative to the substrate in the ground state. To understand the origins of this binding discrimination, we determined the X-ray crystal structures of wild-type MR complexed with two analogues of the putative aci-carboxylate intermediate, benzohydroxamate and Cupferron, to 2.2-Å resolution. Benzohydroxamate is shown to be a reasonable mimic of the transition state and/or intermediate because its binding affinity for 21 MR variants correlates well with changes in the free energy of transition state stabilization afforded by these variants. Both benzohydroxamate and Cupferron chelate the active site divalent metal ion and are bound in a conformation with the phenyl ring coplanar with the hydroxamate and diazeniumdiolate moieties, respectively. Structural overlays of MR complexed with benzohydroxamate, Cupferron, and the ground state analogue (S)-atrolactate reveal that the para carbon of the substrate phenyl ring moves by 0.8-1.2 Å between the ground state and intermediate state, consistent with the proposal that the phenyl ring moves during MR catalysis while the polar groups remain relatively fixed. Although the overall protein structure of MR with bound intermediate analogues is very similar to that of MR with bound (S)-atrolactate, the intermediate-Mg(2+) distance becomes shorter, suggesting a tighter complex with the catalytic Mg(2+). In addition, Tyr 54 moves closer to the phenyl ring of the bound intermediate analogues, contributing to an overall constriction of the active site cavity. However, site-directed mutagenesis experiments revealed that the role of Tyr 54 in MR catalysis is relatively minor, suggesting that alterations in enzyme structure that contribute to discrimination between the altered substrate in the transition state and the ground state by this proficient enzyme are extremely subtle.

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Substrate analogues as probes of the catalytic mechanism of L-mandelate dehydrogenase from Rhodotorula graminis.

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