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Some properties of mandelate racemase from Pseudomonas fluorescens.

ABSTRACT: 1. l-Mandelate dehydrogenase and mandelate racemase were partially purified from extracts of Pseudomonas fluorescens A-312 grown in media containing d-mandelate. 2. The activity of mandelate racemase, but not that of l-mandelate dehydrogenase, is greatly stimulated by Mg(2+), Mn(2+), Co(2+) and, though less effectively, by Ni(2+). Other metal ions are inactive or inhibitory. 3. Racemase activity is inhibited by phosphate, fluoride, pyrophosphate and EDTA. The inhibitions by pyrophosphate and EDTA are competitive with respect to the metal ion activator; those by phosphate and fluoride are competitive with respect to the substrate. 4. The addition of Mg(2+) diminishes the Michaelis constant of racemase. 5. The pH optimum of the racemase is at 7.8. The pH-activity curve of the dehydrogenase complex of enzymes has two peaks, at 7.0 and 8.2. 6. The enzymic racemization of d-mandelate is initially faster than that of l-mandelate. 7. The rates of oxidation of related substrates, catalysed by l-mandelate dehydrogenase, are in the decreasing order: l-p-hydroxymandelate; l-3,4-dihydroxymandelate; l-4-hydroxy-3-methoxymandelate. The racemase is active towards d-p-hydroxymandelate but inactive towards d-3,4-dihydroxymandelate and d-4-hydroxy-3-methoxymandelate. Since 4-hydroxy-3-methoxymandelate, and presumably also 3,4-dihydroxymandelate, arising from the metabolism of catechol-amines, have the d-configuration, the enzymes studied cannot be utilized for estimation of the last two acids in urine.

SUBMITTER: Weil-Malherbe H

PROVIDER: S-EPMC1270079 | biostudies-other | 1966 Oct

REPOSITORIES: biostudies-other

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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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Some properties of mandelate racemase from Pseudomonas fluorescens.

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