The Effect of Aliphatic Alcohols and Organic Solvents on Reactions Catalyzed by 5-Hydroxy-N-methylpyroglutamate Synthetase

Abstract

Abstract 5-Hydroxy-N-methylpyroglutamate synthetase (HMPG synthetase) catalyzes hydrolysis and acyl transfer reactions with δ-substituted α-ketoglutarates, presumably through the formation of an α-ketoglutaryl enzyme intermediate. With α-ketoglutaramate as the substrate, maximal velocities of 18, 26, and 36 are observed for acyl transfer to water, methylamine, and ethanolamine, respectively. The corresponding maximal velocities with ethyl α-ketoglutarate as the acyl donor are 19, 38, and 68. These results suggest that both acylation and deacylation are partially rate limiting for reactions involving α-ketoglutaramate and ethyl α-ketoglutarate. Aliphatic alcohols increase the rate of hydrolysis of α-ketoglutaramate and ethyl α-ketoglutarate. These rate accelerations increase with increasing chain length of the alcohol until a maximal value is reached and are interpreted as a specific effect on the rate of deacylation of the acyl enzyme. Based on this assumption, the rate of transfer of the acyl group from the enzyme to water, to methylamine, and to ethanolamine has been calculated and found to be independent on the nature of the acyl donor. In contrast to the effects observed on hydrolysis, aliphatic alcohols act as competitive inhibitors with respect to amines in acyl transfer reactions and thus provide evidence for an amine-binding site on the enzyme. Organic solvents increase both hydrolysis and transfer reactions involving amines; however, these effects can be distinguished from those of alcohols by several kinetic criteria.