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

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 Kappm, kappcat, 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 (K-1) 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.