FLEXIBILITY OF ENZYMES SUSPENDED IN ORGANIC-SOLVENTS PROBED BY TIME-RESOLVED FLUORESCENCE ANISOTROPY - EVIDENCE THAT ENZYME-ACTIVITY AND ENANTIOSELECTIVITY ARE DIRECTLY RELATED TO ENZYME FLEXIBILITY

A time-resolved fluorescence anisotropy study on the molecular flexibi lity of active-site labeled anthraniloyl-alpha-chymotrypsin, dansylsub tilisin Carlsberg, and native subtilisin Carlsberg, suspended in organ ic solvents, is described. The internal rotational mobility of the flu orophore in the nanosecond time range could be separated from rotation of enzyme aggregates and rapid energy transfer processes. The enzymes suspended in dry organic solvents are less flexible than when dissolv ed in water. The enzyme flexibility increased with increasing hydratio n level. The results confirm that the increase in enzyme activity obse rved upon addition of low amounts of extra water is related to an incr ease in enzyme flexibility. Differences in enantioselectivity of subti lisin Carlsberg in different organic solvents have been correlated wit h differences in enzyme flexibility. The relationship between the inte rnal rotational mobility of the fluorophore and the enantioselectivity provides the first experimental evidence that enzyme flexibility and enzyme enantioselectivity are correlated.