The resulting three-dimensional model of the lipase (Figure 3) showed an extre-
mely high degree of homology with the similarly 1,3-specific lipase from the related
organismRhizomucor miehei. Both enzymes are monomeric proteins, globular in
shape, and possess three disulfide bonds. Examination of theRhizomucor miehei
(Rm) lipase model, and determination of its structure following the binding of an
active site-directed inhibitor, identified a serine (Ser)-histidine (His)-aspartic acid
(Asp) triad that served as the catalytic center, an oxyanion hole appropriately posi-
tioned to stabilize tetrahedral intermediates formed during catalysis, and a hydro-
phobic channel postulated to be the substrate binding region (Brady et al., 1990;
Brzozowski et al., 1991). Analogous structures were identified in the Rd lipase mod-
el (Derewenda et al., 1994a,b). Studies of the Rm lipase model had also identified a
surface peptide loop that covered the active site and appeared to prevent access of
substrate (Brady et al., 1990). Inhibitor studies indicated that, in the presence of
substrate, a physical movement of this ‘lid’ occurred, exposing the active site to
74 4 Cloning, Mutagenesis, and Biochemical Properties
Figure 3. Three-dimensional structure of theRhizopus delemarlipase, determined by X-ray crystallo-
graphy (Derewenda et al., 1994a,b,c; Swenson et al., 1994). (A) The ‘lid’, residues 86–92, is in the closed
conformation and occludes the active site. (B) Lid is partially opened, opening the active site for access by
substrates to the catalytic triad residues: Ser145, Asp204, and His257. (See color plate, page XVII).