5-Hydroxy-N-methylpyroglutamate Synthetase

The enzyme catalyzing the formation of 5-hydroxy-N-methylpyroglutamate has been purified. This enzyme is inactivated by a number of sulfhydryl reagents; however, this inactivation can be prevented by 0.10 m α-ketoglutarate, but not by 0.20 m methylamine. The enzymatic reaction is specific for α-ketoglutarate but a number of primary amines can replace methylamine as substrates for the enzyme. The enzyme-catalyzed hydrolysis of 5-hydroxy-N-methylpyroglutamate is proportional to enzyme over a very narrow range of protein concentration, and the amount of enzyme required to saturate the system is dependent on the substrate concentration. These results are consistent with the hypothesis that N-methyl-α-ketoglutaramate, rather than 5-hydroxy-N-methylpyroglutamate, is the true product of the enzymatic reaction. Measurements of 5-hydroxy-N-methylpyroglutamate formation in the presence of n-propylamine or ethylamine result in alternate substrate inhibition patterns which are consistent with an ordered sequential kinetic mechanism. The enzyme-catalyzed hydrolyses of N-methyl-α-ketoglutaramate and a substrate analogue, α-ketoglutaramate, are noncompetitively inhibited by methylamine when α-keto-glutarate release is used to measure these reactions. However, no inhibition is observed if ammonia release is used as an assay. The decrease in α-ketoglutarate release can be quantitatively accounted for by the formation of 5-hydroxy-N-methylpyroglutamate. These results are consistent with the rate-limiting formation of an α-ketoglutaryl enzyme intermediate.