Kinetic salting effect as a promising tool in the investigation of enzyme molecule changes upon reaction: Deacylation of acyl-chymotrypsins

Abstract

Salting-out (salting-in) effects in the kinetics of enzymatic reactions are discussed. The importance of careful consideration of these effects in p K a 's and other equilibrium constants is stressed. Particular attention is given to the kinetic salting effect which reflects enzyme molecule changes upon reaction that affect interactions of the molecule with the surrounding medium. In acyl-chymotrypsin deacylation, the kinetic salting effect depends on whether the substrate acyl group is situated in the substrate binding center (the hydrophobic slit) or not. A new effect, the salt-dependent promotion, has been found; it is caused by a change in the kinetic salting effect upon modifier binding: the bound modifier mimics the substrate acyl group in the hydrophobic slit. These results are explained by the hypothesis that the hydrophobic slit is compressed in the transition state. The observed salt effect also depends on pH. This is explained by salt dependence of p K a 's and individual rate constants. In the neutral pH region the kinetic salting effect, as well as the salting effect in the catalytic center deprotonation constant, K a1 , must be considered. At high pH's a conformational change occurs: the group with p K a 8.8 in the free enzyme displays in acyl-chymotrypsins p K a values above 11.