A THEORETICAL-STUDY OF THE ACTIVE-SITE OF ENDOTHIAPEPSIN AND 2 MUTANTENZYMES (S35A AND T218A) - IMPLICATIONS FOR CATALYSIS

We have performed ab initio Hartree-Fock calculations on the active si te of endothiapepsin and two ''mutants'' S35A and T218A. The active si te which carries a formal negative charge to effect hydrolysis was mod elled as a formic acid/formate anion moiety and a water molecule. The four nearest hydrogen bonding residues (Gly34, Ser35, Gly217 and Thr21 8) were modelled as formamide and methanol molecules. The most stable active site configuration for wild-type endothiapepsin and the two mut ants has the water molecule bound across the shortest O(D32)...O(D215) diagonal. The inner oxygen of D32 is protonated and the lone pairs of the water oxygen point away from the active site cleft. In this confi guration the water molecule would be expected to be catalytically iner t suggesting that a prototropic rearrangement of the active site must occur during substrate binding to enable the water molecule to attack the scissile bond.