Purification and properties of arogenate dehydrogenase from Actinoplanes missouriensis.

Abstract

Actinoplanes missouriensis utilizes arogenate as an intermediate in L-tyrosine biosynthesis, while no evidence of prephenate dehydrogenase was observed. Arogenate dehydrogenase has been partially purified by a five-step procedure. The enzyme requires NAD as cofactor. The Km values for NAD and arogenate are 0.2 mM and 0.15 mM, respectively. The molecular weight of arogenate dehydrogenase is about 68,000, and SDS gel electrophoresis indicates a composition of two identical subunits. The enzyme is not feedback inhibited by L-tyrosine and unaffected by L-phenylalanine, prephenate, phenylpyruvate, p-hydroxyphenylpyruvate or L-tryptophan. Arogenate dehydrogenase is quite sensitive to p-hydroxymercuribenzoate with 50% inhibition at 12.5 microM of the SH-specific reagent. The presence of malate in usually applied arogenate preparations is demonstrated and the consequence of an impure substrate on arogenate dehydrogenase studies is discussed.