Biosynthesis of leupeptin. III. Isolation and properties of an enzyme synthesizing acetyl-L-leucine.

An enzyme which catalyzes acetylation of L-leucine with acetyl-CoA was partially purified from a cell extract of Streptomyces roseus MA839-A1, a leupeptin producer. The molecular weight of this enzyme is about 27,000 daltons. The enzyme has fairly broad specificity for acyl donors (I) and acceptors (II): as for I, propionyl-CoA was 1/10 as active as acetyl-CoA with L-leucine as the acceptor; as for II, L-leucyl-D-leucine, L-leucyl-L-leucine, L-arginine, L-leucyl-L-leucyl-L-leucine, L-phenylalanine, L-methionine, L-leucine, glycyl-L-phenylalanine and L-valine were acetylated in the decreasing order, as opposed to no or slight reactivity of D-phenylalanine, D-leucine, L-histidine, glycine, L-proline and L-glutamic acid. The Michaelis constants of acetyl-CoA and L-leucine were about 5 X 10(-6) M and 6 X 10(-5) M, respectively. The enzyme is stimulated by Fe++. p-Chloromercuribenzoic acid (PCMB), N-ethylmaleimide, Mg++ and Mn++ were inhibitory. A leupeptin-nonproducing mutant, Streptomyces roseus MA839-A1 LN-S, derived from the producer strain by acriflavine treatment, also produced this enzyme.