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.

Fluorescence Assay for the Binding of Ribonuclease A to the Ribonuclease Inhibitor Protein

“…During their folding and purification, proteins were kept under Ar(g) to prevent oxidation of Cys19. After purification, the thiol of Cys19 was protected from inadvertent air oxidation by reaction with 5,5Ј-dithiobis(2-nitrobenzoic acid) (Sigma Chemical) (32,33). The protected protein was purified from unprotected protein by cation-exchange chromatography.…”

Kinetics and mechanism of the reduction of n-(2-hydroxyethyl)ethylenediaminetriacetatoiron(III) complex by thioglycol in bicarbonate buffer medium

“…The reactions of this substrate have been of interest. The review of its oxidations with different substrates (Chakraborty and Banerjee, 2014; Chakraborty et al, 2012; Shanmugaprabha et al, 2016; Chakraborty et al, 2013; Goswami et al, 2003) and its dimerization to form disulfide in biological systems have been studied (Messmore et al, 2000). Equally, kinetics study on the reduction of Fe(III) aminocarboxylates complexes (Wang et al, 2008;Balahura and Johnson, 1987;Buettner et al, 1983; Xiao-juan et al, 2011; Francis et al, 1985;Bull et al, 1983;Suchecki et al, 2014;Mshelia et al, 2014; Dellert-Ritter and Eldik, 1992) by various reducing substrates have been scantly recorded.…”

Kinetics and Mechanism of Oxidation of 2‐Mercaptoethanol by the Heteropolyoxovanadate [MnV13O38]7−

“…Aliphatic thiols like 2‐mercaptoethanol are of utmost biological importance. The thiol/disulfide couple acts as a buffer in modulating activity of biological sites . The change in the redox potential of the thiol/disulfide couple can usher changes in protein conformation , enzymatic activity, and transport activity.…”