Biosynthesis of leupeptin. II. Purification and properties of leupeptin acid synthetase.

An enzyme which condenses acetyl-L-leucyl-L-leucine and L-arginine into acetyl-L-leucyl-L-leucyl-L-leucyl-L-arginine (leupeptin acid) was partially purified from a cell extract of Streptomyces roseus MA839-A1. With respect to this catalytic activity, the enzyme showed the following characteristics: ATP is essential; optimum pH is 9.5; the activity is inhibited either by EDTA or pyrophosphate or N-ethylmaleimide. The molecular weight of the enzyme is about 260,000 daltons. It also catalyzes some other extension reactions, such as, acetyl-L-leucine+L-leucine+L-arginine leads to leupeptin acid, and acetyl-L-leucine+L-leucine leads to acetyl-L-leucyl-L-leucine, but neither L-leucine+L-arginine leads to (L-leucyl)1--2-L-argining, nor acetyl-L-leucine+L-arginine leads to acetyl-L-leucyl-L-arginine. ATP-PPi exchange, catalyzed by this enzyme, proceeds with either acetyl-L-leucine, or acetyl-L-leucyl-L-leucine or L-leucine, but not with acetate or arginine.