STRUCTURE AND FUNCTION OF SUBTILISIN BPN' SOLUBILIZED IN ORGANIC-SOLVENTS

Enzyme structure and function have been studied for subtilisin BPN' so lubilized in organic solvents by ion pairing with low concentrations o f an anionic surfactant (Aerosol OT) in the absence of reversed micell es. Soluble subtilisin shows strikingly different behavior in octane a nd tetrahydrofuran (THF). In octane, the k(cat)/K-m for the transester ification of N-acetyl-L-phenylalanine ethyl ester (APEE) is 370 M(-1) s(-1), within one order of magnitude of the enzyme's hydrolytic activi ty in water. Moreover, the observed half-life of the soluble enzyme in octane is nearly three orders of magnitude greater than in water, pre sumably because of the absence of autolysis in the organic solvent. In contrast, the catalytic efficiency of the enzyme dissolved in the pol ar solvent THF is 0.04 M(-1) s(-1), and the enzyme loses 99% of its ac tivity within 10 min. Comparable enzyme inactivation could also be obs erved in octane, but only at elevated temperatures such as 70 degrees C. Therefore, the mechanisms of deactivation of the soluble enzyme wer e investigated in both octane and THF. Kinetic and spectroscopic (CD a nd EPR) studies support the existence of multiple inactive forms of th e soluble enzyme in THF at 25 degrees C and in octane at 70 degrees C. Notably, in both cases a denatured form can be renatured in anhydrous octane at 25 degrees C, the first demonstration of enzyme renaturatio n in a bulk organic solvent. A model explaining the THF- and thermally -induced inactivation processes of soluble subtilisin BPN' is proposed , and the apparent reasons for the exceptionally high activity and sta bility of the soluble enzyme in octane are discussed.