N-methylglutamate dehydrogenase: Kinetic studies on the solubilized enzyme

Abstract

N-methylglutamate dehydrogenase has been “solubilized,” by treatment with Triton X-100, and purified about fivefold. Eight different N-methyl amino acids serve as substrates for the enzyme with K m varying from approximately 0.2 m for the poorest substrate, sarcosine, to 54 μ m for the best substrate, N-methyl l-glutamate. The maximal velocity is independent of the N-methyl amino acid, yet decreases more than tenfold when the electron acceptor is changed from phenazine methosulfate to potassium ferricyanide. Double reciprocal plots measuring the effect of 2,6-dichloro-phenolindophenol on the K m of N-methyl l-glutamate show a series of parallel lines. The same result was obtained when sarcosine was used in place of N-methyl l-glutamate. These results are interpreted in terms of a two-step mechanism involving a reduced enzyme intermediate. The lack of product inhibition by glutamate or formaldehyde (or an equimolar mixture of the two) is discussed in terms of the possibility that N-methylene glutamate rather than glutamate or formaldehyde is the true product of the reaction.