Comparison of the kinetics and mechanism of the papain-catalyzed hydrolysis of esters and thiono esters

Abstract

The kinetic constants for the papain-catalyzed hydrolysis of the methyl thiono esters of N-benzoylglycine and N-(beta-phenylpropionyl)glycine are compared with those for the corresponding methyl ester substrates. The k2/Ks values for the thiono esters are 2-3 times higher than those for the esters, and both show bell-shaped pH dependencies with similar pKa's (approximately 4 and 9). The k3 values for the thiono esters are 30-60 times less than those for the esters and do not exhibit a pH dependency. Solvent deuterium isotope effects on k2/Ks and k3 were measured for the ester and thiono ester substrates of both glycine derivatives. Each thiono ester substrate showed an isotope effect similar to that for the corresponding ester substrate. Moreover, use of the proton inventory technique indicated that, as for esters, one proton is transferred in the transition state for deacylation during reactions involving thiono esters and the degree of heavy atom reorganization in the transition state is very similar in both cases. The k3 values for the hydrolysis of a series of para-substituted N-benzoylglycine esters were found to correlate with the k3 values for the corresponding para-substituted thiono esters [Carey, P. R., Lee, H., Ozaki, Y., & Storer, A. C. (1984) J. Am. Chem. Soc. 106, 8258-8262], showing that the rate-determining step for the deacylation of both thiolacyl and dithioacyl enzymes probably involves the disruption of a contact between the substrate's glycinic nitrogen atom and the sulfur of cysteine-25. It is concluded that the hydrolysis of esters and thiono esters proceeds by essentially the same reaction pathway. Due to an oxygen-sulfur exchange process the product released in the case of the N-(beta-phenylpropionyl)glycine thiono ester substrate is the dioxygen acid; however, for the N-benzoylglycine thiono ester substrate, the thiol acid is the initial product. This thiol acid then acts as a substrate for papain and reacylates the enzyme to eventually give the dioxygen acid product. It is shown that thiol acids are excellent substrates for papain.

Role of the Carbonyl Group in Thioester Chain Length Recognition by the Medium Chain Acyl-CoA Dehydrogenase

“…Comparison of these structures showed that the 2'-OH-of the ribityl side chain of FAD would have to be rotated away from the carbonyl oxygen of the substrate to accommodate the larger sulfur atom. While some enzymes can tolerate such substitution with little effect (Storer & Carey, 1985), replacement of C=O by C=S generally significantly slows catalysis [e.g., Wlassics et al (1988) and Anderson et al (1990)l. Thus, the comparatively weak binding of 2-azadithiooctanoyl-CoA, the low turnover with dithiooctanoyl-CoA, and the molecular modeling results are consistent with a tight interaction between the substrate carbonyl oxygen and its binding pocket in the medium chain dehydrogenase.…”

A Reexamination of the Substrate Utilization of 2-Thioorotidine-5′-monophosphate by Yeast Orotidine-5′-Monophosphate Decarboxylase

“…Additionally, Miller et al (21) have shown that removal of the Gln215 side chain, the only active-site functional group within proximity of O2 of the bound ligand, is inconsequential for decarboxylase activity. It is predictable from these observations that 2-thioUMP would have approximately the same affinity as UMP, as there appears to be ample accommodation for the larger CϭS group, which is typically 0.4 Å longer than a CϭO double bond (22). It is not evident, however, why 2-thioOMP does not bind and undergo decarboxylation if it occupies the same orientation as UMP as seen in the crystal structures, and that projected for OMP by the mechanism in Fig.…”

Utility of Acetyldithio-CoA in Detecting the Influence of Active Site Residues on Substrate Enolization by 3-Hydroxyl-3-methylglutaryl-CoA Synthase

“…Collapse of the tetrahedral intermediate formed after solvent attack will reform the covalent adducts but may result in loss of sulfur since this would be a good leaving group (20). Such sulfur/oxygen exchange from an enzyme-dithio-HMG-CoA adduct, followed by cleavage of the covalent linkage to enzyme, would produce unsubstituted HMGCoA.…”