CONTRIBUTION OF CUTINASE SERINE-42 SIDE-CHAIN TO THE STABILIZATION OFTHE OXYANION TRANSITION STATE

Cutinase from the fungus Fusarium solani pisi is a lipolytic enzyme ab le to hydrolyze both aggregated and soluble substrates. It therefore p rovides a powerful tool for probing the mechanisms underlying lipid hy drolysis. Lipolytic enzymes have a catalytic machinery similar to thos e present in serine proteinases. It is characterized by the triad Ser, His, and Asp (Glu) residues, by an oxyanion binding site that stabili zes the transition state via hydrogen bonds with two main chain amide groups, and possibly by other determinants. It has been suggested on t he basis of a covalently bond inhibitor that the cutinase oxyanion hol e may consist not only of two main chain amide groups but also of the Ser42 O gamma side chain. Among the esterases and the serine and the c ysteine proteases, only Streptomyces scabies esterase, subtilisin, and papain, respectively, have a side chain residue which is involved in the oxyanion hole formation. The position of the cutinase Ser42 side c hain is structurally conserved in Rhizomucor miehei lipase with Ser82 O gamma, in Rhizopus delemar lipase with Thr83 O gamma 1, and in Candi da antartica B lipase with Thr40 O gamma 1. To evaluate the increase i n the tetrahedral intermediate stability provided by Ser42 O gamma 1 w e mutated Ser42 into Ala. Furthermore, since the proper orientation of Ser42 O gamma is directed by Asn84, we mutated Asn84 into Ala, Leu, A sp, and Trp, respectively, to investigate the contribution of this ind irect interaction to the stabilization of the oxyanion hole. The S42A mutation resulted in a drastic decrease in the activity (450-fold) wit hout significantly perturbing the three-dimensional structure. The N84 A and N84L mutations had milder kinetic effects and did not disrupt th e structure of the active site, whereas the N84W and N84D mutations ab olished the enzymatic activity due to drastic steric and electrostatic effects, respectively.