Sulfur shuffle: modulating enzymatic activity by thiol-disulfide interchange.

Abstract

The facile modulation of biological processes is an important goal of biological chemists. Here, a general strategy is presented for controlling the catalytic activity of an enzyme. This strategy is demonstrated with ribonuclease A (RNase A), which catalyzes the cleavage of RNA. The side-chain amino group of Lys41 donates a hydrogen bond to a nonbridging oxygen in the transition state for RNA cleavage. Replacing Lys41 with a cysteine residue is known to decrease the value of k(cat)/K(m) by 10(5)-fold. Forming a mixed disulfide between the side chain of Cys41 of K41C RNase A and cysteamine replaces the amino group and increases k(cat)/K(m) by 10(3)-fold. This enzyme, which contains a mixed disulfide, is readily deactivated by dithiothreitol. Forming a mixed disulfide between the side chain of Cys41 and mercaptopropyl phosphate, which is designed to place a phosphoryl group in the active site, decreases activity by an additional 25-fold. This enzyme, which also contains a mixed disulfide, is reactivated in the presence of dithiothreitol and inorganic phosphate (which displaces the pendant phosphoryl group from the active site). An analogous control mechanism could be installed into the active site of virtually any enzyme by replacing an essential residue with a cysteine and elaborating the side chain of that cysteine into appropriate mixed disulfides.