Enzymology in Aqueous-Organic Cosolvent Binary Mixtures

The theory and results of solvent structure control on solute behavior for ordinary chemical reactions, generated in other laboratories (e.g. by Arnett and colleagues), were applied to enzyme reactions in water-organic cosolvent binary mixtures. Three kinds of reactions were studied: (a) catalysis of substrate reactions; (b) denaturation, judged by loss of overall activity; (c) renaturation, aided by organic cosolvents after heat denaturation in water, judged by relative activity. The enzymes studied were trypsin, potato acid phosphatase, l-glutamate dehydrogenase, bacterial β-glucuronidase, glucose 6-phosphate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, and lysozyme. In the critical 0.01 to 0.05 mole fraction region, with four carbon alcohols and sometimes dioxane, the results generally were: (a) the enzymes are slightly (a factor of 1½ to 3 times) more active than they are in water only. Occasionally they are markedly more active (factor of 10 or more). In no case are they less active in the binary mixture than in water unless the critical mole fraction region is exceeded. (b) The cosolvents enhance the tendency to heat denature. (c) They also enhance the tendency to renature at lower temperatures after heating in neat water. As expected from the general results of other laboratories in physical organic chemistry, the lower alcohols and ethylene glycol are much less effective than the higher alcohols in the critical mole fraction ranges. The design of experiments with enzymes and interpretation of the results hinge on the conformationally motile model in all phenomena. There is a good possibility that in vivo enzymes, which in effect are in solvents other than neat water, are more active than they are n strictly aqueous in vitro systems.