Effects of anions on the activation thermodynamics and fluorescence emission spectrum of alkaline phosphatase: evidence for enzyme hydration changes during catalysis

Abstract

The effects of anions on the thermodynamic activation functions for a model enzyme, calf intestinal alkaline phosphatase were studied in order to examine the role of protein hydration changes in establishing the energetics of enzyme catalysis. The influences of these anions on the activation volume .**GRAPHIC**. and activation free energy .**GRAPHIC**. reflected clear Hofmeister (lyotropic) series effects, in the order F- > Cl- > Br- > I- (order of increasing salting-out potential). A pronounced covariation was observed between the influences of these anions on Vmax, which is proportional to .**GRAPHIC**. and on the negative activation volume of the reaction. Fluoride was able to counteract the influences of Br- and I- on both Vmax and .**GRAPHIC**. when combinations of these anions were employed. The effects of Br- and I- on Vmax and .**GRAPHIC**. were more pronounced at lower temperatures. The control .**GRAPHIC**. was increasingly negative at reduced temperatures. The effects of the neutral salts and propanol on .**GRAPHIC**. and .**GRAPHIC**. as well as the effects of salting-in anions on the activation enthalpy and the negative activation entropy of the reaction, are consistent with a model which proposes that peptide groups or polar side chains on the native enzyme exergonically increase their exposure to solvent during the catalytic activation event. These conclusions are in accord with the known free energy, enthalphy, entropy and volume changes which occur when model peptide groups are transferred between water and concentrated salt solutions. Consistent with the kinetic results, the fluorescence emission wavelength maximum of alkaline phosphatase increased in the presence of anions in the order F- > Cl- > Br- > I-. The salting-out ion (F-) and the salting-in ions (Br- and I-) shifted .lambda.max in different directions, and these .lambda.max shifts could be counterbalanced by using equimolar combinations of salting-in and salting-out anions. Control experiments with a model compound, N-acetyltryptophanamide, showed that the spectral shifts caused by the salts did not result solely from differential quenching by the anions of the solvent-exposed tryptophan(s) on the enzyme. Hofmeister additivity phenomena indicated that the solvent is at the basis of these salt-induced enzyme structural changes. Changes in protein solvation during enzymic reactions contribute significantly to the thermodynamic activation parameters in both the native and the salt-perturbed enzyme.