alpha-Chymotrypsin stability in aqueous-acetonitrile mixtures: is the native enzyme thermodynamically or kinetically stable under low water conditions?

Like many proteins, alpha-chymotrypsin is denatured in 50% volume aqueous-a cetonitrile mixtures. However, it also shows high catalytic activity in 70% or more acetonitrile. Good activity in two different aqueous organic compo sition ranges has been described for several other enzymes. The stability o f the: native protein under low water conditions is generally believed to b e a kinetic phenomenon, though there are also arguments for thermodynamic s tability. We have distinguished between these possibilities by studying the effects of changing medium composition at different times. In preliminary experiments, we found catalytic activity could be recovered by adding neat acetonitrile to chymotrypsin in a 50% mixture, suggesting that the enzyme c ould renature under these conditions. However, in the 50% mixture, the true initial activity at 30 degrees C is not zero, as the literature suggests. Instead, there is an initial burst of product formation over a. few minutes , after which the enzyme becomes inactivated. By pre-incubating a 50% aqueo us-acetonitrile mixture at 30 degrees C prior to enzyme addition, the produ ct burst could be eliminated. Activity could not then be recovered by slow addition of acetonitrile to the denatured enzyme. In contrast, it was possi ble to renature by dilution with aqueous buffer so that regeneration of cat alytic activity was achieved. Thus, the good practical performance at high acetonitrile concentrations almost certainly results from a high kinetic ba rrier towards denaturation. The kinetics of enzyme denaturation in 50% and 70% acetonitrile were also investigated both at 30 and 20 degrees C. Loss o f catalytic activity was faster at higher temperature and at lower acetonit rile concentrations. (C) 1999 Elsevier Science B.V. All rights reserved.