Molecular dynamics of microbial lipases as determined from their intrinsictryptophan fluorescence

We have studied the intrinsic tryptophan fluorescence of the lipases from C hromobacterium viscosum (CVL), Pseudomonas species (PSL), and Rhizopus oryz ae (ROL) in aqueous buffer, zwitterionic detergent micelles, and isopropano l-water mixtures. It was the purpose of this study to obtain information ab out biophysical properties of the respective enzymes under conditions that modulate enzyme activities and stereoselectivities to a significant extent. According to their decay-associated emission spectra, CVL tryptophans are located in the hydrophobic interior of the protein. in contrast, the PSL an d ROL tryptophans are probably confined to the core and the surface of the lipase. From the tryptophan lifetime distributions it can be concluded that the conformation of CVL is not much affected by detergent or organic solve nt (isopropanol). Accordingly, CVL is enzymatically active in these systems and most active in the presence of isopropanol. In contrast, ROL and PSL s how high conformational mobility, depending on the solvent, because their l ifetime distributions are very different in the presence and absence of det ergent or isopropanol. Time-resolved anisotropy studies provided evidence t hat the lipases exhibit very high internal molecular flexibility. This pecu liar feature of lipases is perhaps the key to the great differences in acti vity and stereoselectivity observed in different reaction media. Furthermor e, information about self-association of the lipases in different solvents could be obtained. PSL, but not CVL and ROL, forms aggregates in water. Lip ase aggregation can be reversed by the addition of detergent or isopropanol , which competes for the hydrophobic surface domains of this protein. This dissociation could efficiently contribute to the increase in lipase activit y in the presence of a detergent or isopropanol.