Catalytic properties of the metal ion variants of mandelate racemase reveal alterations in the apparent electrophilicity of the metal cofactor

Abstract

Mandalate racemase (MR) from Pseudomonas putida requires a divalent metal cation, usually Mg²⁺, to catalyse the interconversion of the enantiomers of mandelate. Although the active site Mg²⁺ may be replaced by Mn²⁺, Co²⁺, or Ni²⁺, substitution by these metal ions does not markedly (<10-fold) alter the kinetic parameters K^app_m, k^app_cat, and (k_cat/K_m)^app for the substrates (R)- and (S)-mandelate, and the alternative substrate (S)-trifluorolactate. Viscosity variation experiments with Mn²⁺-MR showed that the metal ion plays a role in the uniform binding of the transition states for enzyme–substrate association, the chemical step, and enzyme-product dissociation. Surprisingly, the competitive inhibition constants (K_i) for inhibition of each metalloenzyme variant by benzohydroxamate did not vary significantly with the identity of the metal ion unlike the marked variation of the stability constants (K₁) observed for M²⁺·BzH complex formation in solution. A similar trend was observed for the inhibition of the metalloenzyme variants by F⁻, except for Mg²⁺-MR, which bound F⁻ tighter than would be predicted based on the stability constants for formation of M²⁺·F⁻ complexes in solution. Thus, the enzyme modifies the enatic state of the bound metal ion cofactor so that the apparent electrophilicity of Mg²⁺ is enhanced, while that of Ni²⁺ is attenuated, resulting in a levelling effect relative to the trends observed for the free metals in solution.