Reactivity of trypsin in reverse micelles: pH-effects on the W-0 versus enzyme activity profiles

pH-Dependence of hydrolytic activity of trypsin has been studied in cationi c reverse micellar system of cetyltrimethylammonium bromide (CTAB) in (50% v/v) chloroform/isooctane using a positively charged substrate N-alpha-benz oyl-L-arginine ethyl ester (BAEE). The pH of the medium was varied from 4.0 to 8.5 with addition of 0.025 M citrate-phosphate buffer containing 1 mM C aCl2. Optimum pH for maximum enzyme activity, pH(opt) in reverse micelles i s found to be similar to that observed in bulk aqueous solution (8.0-8.5). However, changes in activity of trypsin (k(cat)) as a function of water con tent W-0 (W-0 = [H2O]/[CTAB]) in reverse micelles are found to be pH depend ent. At low pH (4.0) and low water content (W-0 = 5) the enzyme is more act ive in reverse micelles than in bulk aqueous solution by a factor of 2. Thi s 'superactivity' is lost at higher W-0 values and the k(cat) in reverse mi celles is found to be similar to that observed in aqueous bulk. At pH 5, th e enzyme activity is found to be independent of W-0 while at pH 6.0-6.5 the enzyme activity is low at W-0 5 and increases with water content to a cons tant value which is still 50% lower than that in aqueous buffer. Above pH 7 , the W-0-activity profile becomes distinctly bell shaped with W-0 optimum around 10-15. The enzyme activity at optimum W-0 is close to that observed in aqueous bulk. (C) Societe francaise de biochimie et biologie moleculaire / Elsevier, Paris.