THE ENHANCING OF A CYSTEINE PROTEINASE ACTIVITY AT ACIDIC PH BY PROTEIN ENGINEERING, THE ROLE OF GLUTAMIC-50 IN THE ENZYME MECHANISM OF CARICAIN

Carica papaya produces four cysteine proteinases, Calculations show th at the Cys(25), His(159) essential ion pair is fully ionised at pH 2.9 9, where activity cannot be detected, but apparently an additional ion isation with a pK(a) of 4 is essential for activity (an electrostatic switch). Caricain (EC 3.4.22.30) wt and D158E genetic backgrounds were used to study the contribution of E50A to activity. E50 or E135 are c andidates for the switch, E50A would be expected to reduce activity, H owever, activity increased at pH 5.0 in both backgrounds and at the pH optimum in D158E E50A but decreased slightly in the wt background. Th is challenges the hypothesis of an electrostatic switch. (C) 1998 Fede ration of European Biochemical Societies.