Purification and properties of an enzyme reducing leupeptin acid to leupeptin.
An enzyme catalyzing the reduction of leupeptin acid to leupeptin was partially purified from a cell extract of Streptomyces roseus MA839-A1, a leupeptin producer. The enzyme was tentatively named leupeptin acid reductase. The molecular weight was estimated to be 320,000 by chromatography on Sepharose 6B. The reductase eluted with leupeptin acid synthetase both in molecular sieve chromatography and in affinity chromatography. The main properties of the reductase were: (1) ATP and NADPH were required for activity. ATP could not be replaced by GTP, ADP or AMP. NADPH could not be replaced by NADH. (2) Michaelis constants for ATP and NaDPH were 4.2 . 10(-5) M and 1.3 10(-6) M, respectively. (3) The enzyme was inhibited by leupeptin, the reaction product, and antipain. Both inhibitors have an L-argininal residue at the C-terminal structure. (4) The enzyme did not catalyze the conversion of leupeptin to leupeptin acid. Leupeptin acid reductase and leupeptin acid synthetase were found in the 10,000 x g pellet of the cell homogenate. The reductase was not released as readily from the pellet as the synthetase either by washing or by repeated freeze-thawing. Synthesis of leupeptin from acetyl-CoA, L-lucine and L-arginine in vitro was accomplished by combining leucine acyltransferase and the enzyme complex consisting of leupeptin acid synthetase an leupeptin acid reductase.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000445:Aldehyde Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D004355:Drug Stability; D007976:Leupeptins / Q000378:metabolism*; D008970:Molecular Weight; D009249:NADP / Q000494:pharmacology; D009842:Oligopeptides / Q000378:metabolism*; D010084:Oxidation-Reduction; D010453:Peptide Synthases / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology
D007976:Leupeptins; D009842:Oligopeptides; C032853:leupeptin acid; D009249:NADP; D000255:Adenosine Triphosphate; D000445:Aldehyde Oxidoreductases; C032889:leupeptin acid reductase; D010453:Peptide Synthases; C023433:leupeptin acid synthetase; C032854:leupeptin