ADENOSYLCOBALAMIN-DEPENDENT GLUTAMATE MUTASE - EXAMINATION OF SUBSTRATE AND COENZYME BINDING IN AN ENGINEERED FUSION PROTEIN POSSESSING SIMPLIFIED SUBUNIT STRUCTURE AND KINETIC-PROPERTIES

Glutamate mutase is comprised of two weakly associating Subunits, E an d S, that combine to form the coenzyme binding site, The active holoen zyme assembles in a kinetically complex process in which both the stoi chiometry and apparent K-d for adenosylcobalamin (AdoCbl) are dependen t upon the relative concentrations of the two subunits, as is the enzy me's specific activity, To facilitate mechanistic and structural studi es on this enzyme we have genetically fused the S subunit to the C-ter minus of the E subunit through an 11 amino acid (Gly-Gln)(5)-Gly linke r segment. This protein, GlmES, binds AdoCbl stoichiometrically and ne ither the affinity for AdoCbl nor;the turnover number depends upon pro tein concentration, The k(cat) and K-m for both substrate and coenzyme , together with the deuterium isotope effects on V-max and V-max/K-m, have been determined for the GlmES-catalyzed reaction proceeding in bo th directions. Compared with wild type, the affinity for AdoCbl is unc hanged, but for the conversion of L-glutamate to (2S,3S)-3-methylaspar tate both k(cat) and K-m for L-glutamate are decreased by about a thir d and the isotope effects are reduced, suggesting product release to b e more rate-limiting, To test hypotheses concerning the activation of the coenzyme, we examined the binding of adenosylcobalamin, methylcoba lamin, and cob(II)alamin to the enzyme. Each of these is bound with es sentially the same affinity (2 mu M), suggesting that, contrary to exp ectations, interactions between the protein and the adenosyl moiety do not serve to weaken the cobalt-carbon bond in the ground state.