Mechanism of action of adenosylcobalamin: glycerol and other substrate analogs as substrates and inactivators for propanediol dehydratase - kinetics, stereospecificity, and mechanism

Abstract

A number of vicinal diols were found to react with propanediol dehydratase, typically resulting in the conversion of enzyme-bound adenosylcobalamin to cob(Il)alamin and formation of aldehyde or ketone derived from substrate. Moreover, all are capable of effecting the irreversible inactivation of the enzyme. The kinetics and mechanism of product formation and inactivation were investigated. Glycerol, found to be a very good substrate for diol dehydratase as well as a potent inactivator, atypically, did not induce cob(II)alamin formation to any detectable extent. With glycerol, the inactivation process was accompanied by conversion of enzymebound adenosylcobalamin to an alkyl or thiol cobalamin, probably by substitution of an amino acid side chain near the active site for the 5'-deoxy-5'-adenosyl ligand on the cobala-min. The inactivation reaction with glycerol as the inactivator exhibits a deuterium isotope effect of 14, strongly implicating hydrogen transfer as an important step in the mechanism of inactivation. The isotope effect on the rate of product formation was found to be 8.0. Experiments with isotopically substituted glycerols indicate that diol dehydrase distinguishes between "R" and "S" binding conformations, the enzyme-(R)-glycerol complex being predominately responsible for the productforming reaction, while the enzyme-(S)-glycerol complex results primarily in the inactivation reaction. Mechanistic implications are discussed. A method for removing enzymebound hydroxycobalamin that is nondestructive to the enzyme and a technique for measuring the binding constants of (R) and (S)-1,2-propanediols are presented.

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