Binding of competitive (product) inhibitors to papain in the active and inactive state

Abstract

1. 1. In X-ray studies of crystalline papain product complexes, it was found that the active site was never occupied more than 30% with the product. This could be due to blocking of the essential SH in a fraction of the papain molecules while assuming that these non-active enzyme molecules do not bind the product. To prove this hypothesis product-inhibitor experiments were carried out in solution with Boc- p-I-Phe-leu ( K i ∼ 1·10 −6 M at pH 4) and benzoylarginine, with K i ∼ 0.03 M at pH 4. 2. 2. Two experimental approaches were applied. The first approach is applicable, when K i , the dissociation constant of enzyme-product complex, has the same order of magnitude as the free enzyme concentration. In that case the fractional inhibition caused by the product depends on the enzyme concentration. In the second method the alkylation of papain by iodoacetic acid or iodoacetamide is measured as a function of the Boc- p-I-Phe-Leu or the benzoylarginine concentration. 3. 3. The results obtained with both methods indicate that any blocking of the essential SH in papain prevents binding of products. The possibility is discussed that the observed binding constant K i of a product must be ascribed to the formation of the corresponding acylenzyme.