CUTINASE, A LIPOLYTIC ENZYME WITH A PREFORMED OXYANION HOLE

Cutinases, a group of cutin degrading enzymes with molecular masses of around 22-25 kDa (Kolattukudy, 1984), are also able to efficiently hy drolyse triglycerides (De Geus et al., 1989; Lauwereys et al., 1991), but without exhibiting the interfacial activation phenomenom (Sarda et al., 1958). They belong to a class of proteins with a common structur al framework, called the alpha/beta hydrolase fold (Martinez et al., 1 992; Ollis et al., 1992). We describe herein the structure of cutinase covalently inhibited by diethyl-p-nitrophenyl phosphate (E600) and re fined at 1.9-angstrom resolution. Contrary to what has previously been reported with lipases (Brzozowski et al., 1991; Derewenda et al., 199 2; Van Tilbeurgh et al., 1993), no significant structural rearrangemen t was observed here in cutinase upon the inhibitor binding. Moreover, the structure of the active site machinery, consisting of a catalytic triad (S120, H188, D175) and an oxyanion hole (Q121 and S42), was foun d to be identical to that of the native enzyme, whereas the oxyanion h ole of Rhizomucor lipase (Brzozowski et al., 1991; Derewenda et al., 1 992), like that of pancreatic lipase (van Tilbeurgh et al., 1993), is formed only upon enzyme-ligand complex formation. The fact that cutina se does not display interfacial activation cannot therefore only be du e to the absence of a lid but might also be attributable to the presen ce of a preformed oxyanion hole.