Neutral salt effects on the velocity and activation volume of the lactate dehydrogenase reaction: Evidence for enzyme hydration changes during catalysis

Abstract

The effects of different neutral salts on the maximal velocity ( V) and activation volume ( ΔV‡ ) of the M 4-lactate dehydrogenase reaction were studied to determine the mechanistic basis of the inhibitory effects of these salts. For salting-in salts (which increase protein group solubility), increasing salt concentrations led to reductions in V and increases in ΔV‡ , with the order of salt effectiveness following the Hofmeister (lyotropic) series: KSCN > KI > KBr. A 50% reduction in V was associated with an approximately 17 cm 3 mol −1 increase in ΔV‡ for different concentrations of the same salt and for equal concentrations of different salting-in salts. Salting-out salts were also inhibitory, but no uniform correlation between changes in V and ΔV‡ was observed. The strongly salting-out salt KF decreased ΔV‡ at all concentrations. The weaker salting-out salt K 2SO 4 increased ΔV‡ at concentrations below 0.1 m and decreased ΔV‡ at higher concentrations. KCl increased ΔV‡ as the salt concentration was raised to approximately 0.2 m; further increases in KCl concentration were without effect on ΔV‡ . The rate and volume effects of these neutral salts, especially the highly regular covariation in V and ΔV‡ found for salting-in salts, seem difficult to explain in terms of salt-induced changes in the geometry of the active site. We propose instead that these salt effects can all be explained in terms of the energy and volume changes which accompany transfers of protein groups (amino acid side chains and peptide backbone linkages) between the hydrophobic interior of the enzyme and the enzyme-water interface during catalytic conformational changes.