STUDYING THE ROLE OF THE DIVALENT METAL CATION ACTIVATOR OF THE CAMBIALISTIC ENZYME MANDELATE RACEMASE

Abstract

Mandalate racemase (MR) from Pseudomonas putida requires a divalent metal cation, usually Mg2+, to catalyze the interconversion of the enantiomers of mandelate. Typical of a cambialistic enzyme, the active site Mg2+ may be replaced by other metal ions. Replacement by Co2+, Ni2+, and Mn2+ does not significantly alter the kinetic parameters Kmapp, kcatapp, and (kcat/Km)app for the substrates (R)– and (S)–mandelate, and the alternative substrate (S)–trifluorolactate. The competitive inhibition constants (Ki) for inhibition of each metalloenzyme by benzohydroxamate did not vary significantly with the identity of the metal ion; however, marked variation of the stability constant (K1) for metal ion–BzH complex formation in solution was observed. Thus the enzyme appears to modify the properties of the bound metal ion so that the catalytic roles of the different metal ions are relatively invariant compared to their properties in solution (‘Hephaestus effect’). The slight reduction in catalytic efficiency by Mn2+–MR was studied by examining the dependence of kcatapp and (kcat/Km)app for Mn2+–MR on the solvent microviscosity. These studies showed that the metal ion plays a role in the uniform binding of the transition states for substrate/product association/dissociation and the chemical step.