Solvent Isotope Effects on Formation of Protease Complexes with Inhibitory Aldehydes, with an Appendix on the Determination of Deuterium Fractionation Factors by NMR

Article abstract of DOI:10.1021/ja00302a028

In both CCl4 and water, equilibria of addition of thiols to acetaldehyde are strongly favored by the presence of deuterium at exchangeable positions, whereas equilibria of addition of hydroxylic compounds are hardly affected.To test the possibility of using solvent isotope effects for diagnosing the structure of enzyme-inhibitor complexes, we examined the influence of D2O on the interaction of proteases with inhibitory aldehydes.Equilibrium constants for binding of inhibitory aldehydes showed enhancements in D2O of 1.32+/-0.12 for α-chymotrypsin and 2.65+/-0.14 for papain, slightly in excess of those expected for covalent addition of an alcohol or thiol, respectively.In an Appendix, hydroxylic solutes are shown to alter the magnetic susceptibilities of solutions, resulting in systematic errors in apparent fractionation factors of their exchangeable protons as determined by conventional NMR procedures.As a result, apparent fractionation factors change with the orientation of the magnetic field.Corrected values for hydroxylic protons, determined by other methods, differ only slightly from unity.