SUBSTRATE EFFECTS ON THE ENZYMATIC-ACTIVITY OF ALPHA-CHYMOTRYPSIN IN REVERSE MICELLES

Six different substrates have been used for measuring the activity of α-chymotrypsin in reverse micelles formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane. The substrates were glutaryl-Phe p-nitroanilde, succinyl-Phe p-nitroanilide, acetyl-Phe p-nitroanilide, succinyl-Ala-Ala-Phe p-nitroanilide, succinyl-Ala-Ala-Pro-Phe p-nitroanilide and acetyl-Trp methyl ester. It has been shown that the dependence of the kinetic constants (kcat and Km) on the water content of the system, on wo (= [H2O] [AOT]), is different for the different substrates. This indicates that activity-wo profiles for α-chymotrypsin in reverse micelles not only reflect an intrinsic feature of the enzyme alone. For the p-nitroanilides it was found that the lower kcat (and the higher Km) in aqueous solution, the higher kcat as well as Km in reverse micelles. "Superactivity" of α-chymotrypsin could only be found with the ester substrate and with relatively "poor" p-nitroanilides. The presence of a negative charge in the substrate molecule is not a prerequesite for α-chymotrypsin to show "superactivity". © 1991.