THE PAPAIN-CATALYZED SYNTHESIS OF HIPPURYL ANILIDE. I. GENERAL PROPERTIES OF THE ENZYME SYSTEM.

Abstract

1. 1. The ability of purified, crystalline, chymopapain-free papain (EC 3.4.4.10) to synthesize hippuryl anilide from hippuric acid and aniline has been investigated. The kinetic method of determining the reaction velocity by following aniline disappearance has been employed. 2. 2. The hydrolytic and synthetic activities of the enzyme were compared. Using 3. N-benzoyl-l-argininamide, and hippuric acid and aniline, as substrates for the estimation of hydrolytic and synthetic activities, respectively, papain was found to be over 1000 times more efficient in hydrolyzing N-benzoly-l-argininamide than in synthesizing hippuryl anilide. 4. 3. Chymopapain (EC 3.4.4.11), obtained in a partially purified state, was shown to be more active than crystalline papain in promoting the synthesis of hippuryl anilide. 5. 4. Papain is remarkably stable to heat in the absence of activator, but enzymic activity is readily lost in the presence of β-mercaptopropionic acid. Hippuric acid alters the heat stability of inactive papain and its presence enhances the rate at which papain loses its potential to undergo activation by sulfhydryl activator. 6. 5. 100 moles of sulfhydryl activator, β-mercaptopropionic acid, er mole of en enzyme are required for maximal activity. Diethyldisulfide-β,β′-dicarboxylic acid, the oxidation product of β-mercaptopropionic acid, inhibits enzymic synthesis at high concentrations. There is no synthesis in the absence of activator or in the presence of ethylenediaminetetra acetate alone. Ethylenediaminetetraacetate does not enhance the synthetic activity in the presence of sulfhydryl activator and citrate buffer. 7. 6. The velocity of hippuryl anilide synthesis is a linear function of papain concentration up to 6 mg/ml. 8. 7. Papain hydrolyzes the hippuryl anilide slowly due to the low solubility, 1.85 · 10−4 M, in citrate buffer of the anilide.