Analysis of conformational states of Candida rugosa lipase in solution: Implications for mechanism of interfacial activation and separation of open and closed forms

In this study, an analysis of the transition between the inactive ("closed" ) and active ("open") conformations of Candida rugosa lipase in solution is performed using irreversible enzyme inhibitors, cyclic saligenin phosphate s. It is shown that >90% inhibition of the enzyme activity toward water-sol uble substrates (esterolytic activity) can be achieved with as little as 0. 3 mol of the inhibitor per mole of enzyme, whereas activity toward emulsifi ed substrates decreases by approximately 20% under the same conditions. It is also shown that short-term exposure of this inhibited enzyme preparation to an interface leads to a significant increase in esterolytic activity, w hich even exceeds that of the untreated control. These experimental observa tions suggest that the inhibitors interact predominantly, if not exclusivel y, with the open form of the enzyme and that any transitions occurring betw een the two conformers of the enzyme in solution, in the absence of an inte rface, are extremely slow. This conclusion is verified by separating the op en and closed forms of the enzyme by hydrophobic interaction column chromat ography on phenylsepharose. Fractions enriched with the respective conforma tions of the enzyme are further purified using gel-permeation chromatograph y. On the basis of the elution pattern from this step, and sodium dodecylsu lfate-polyacrylamide gel electrophoresis (SDS-PAGE), the open (active in th e absence of interface) form of the lipase is found to be present in soluti on as a dimer, whereas the closed form appears to be a monomer. The latter form of the enzyme may be activated by up to 60-fold when exposed to triole in. (C) 2001 John Wiley & Sons, Inc.