Front Matter

that of its single-mutant progenitors (Klein et al., 1997a). However, not surprisingly,

the introduction of these large amino acid side chains into the substrate binding

trough also significantly reduced the overall activity of the enzyme.

Molecular modeling indicated that the introduction of a salt bridge across the

substrate binding trough slightly farther from the catalytic center might allow access

of mid-chain fatty acids while blocking the entry of longer ones. When the corre-

sponding double mutant was created, a three fold increase in activity toward mid-

chain fatty acids, compared to that toward long-chain substrates, was observed

(Klein et al., 1997a).

One of the most interesting mutant lipases produced during this work was a double

mutant wherein a phenylalanine on one side of the binding site (Phe95) was replaced

by Asp, while a valine on the other side (Val206) was replaced by a threonine (Klein

et al., 1997b). The activity of the wild-type enzyme toward medium chain length

fatty acids was about 1.5 times that toward long-chain fatty acids. Singly, each mu-

tation increased activity toward mid-chain fatty acids to 4-to 6-fold greater than that

toward long-chain ones. In the double mutant this preference increased to more than

7-fold, roughly an addition of the effects of the individual mutations. Also, whereas

the single mutations approximately doubled the activity of the enzyme toward mid-

chain fatty acids relative to that toward short chains, the activity of the double mutant

toward mid-chain fatty acids was about 100-fold greater than that toward short-chain

fatty acids.

Quite unexpectedly, the double mutant also displayed a very sharp and striking

dependence of activity upon pH (Klein et al., 1997b). At pH 7.0 the enzyme dis-

played strong activity and strong selectivity toward mid-chain fatty acids. At,

and below, pH 6.5 activity toward these substrates fell, and that toward short-chain

substrates increased. At pH 7.5 the enzyme was virtually inactive against all sub-

strates. Thus, the enzyme displayed striking substrate selectivity, but only under

a narrow range of conditions. It is unclear whether this behavior is caused by

pH-mediated polarization or ionization of the Asp introduced by mutation.

These experiments began the precise correlation of structure with substrate spe-

cificity in the lipases, emphasized the importance of choice of assay conditions when

screening mutant libraries for activity and specificity, and indicated reaction condi-

tions under which unique substrate selectivity could be achieved. Considerable ad-

ditional work could be conducted in these areas, since the role of enzyme structure in

determining substrate selectivity in the hydrolytic mode has only roughly been iden-

tified, and its impact on substrate selectivity in ester synthesis reactions remains

unexplored.

4.8 Perspective

It is difficult to convey to those coming recently to the study of lipases just how little

was known about these enzymes a mere 15 years ago. At that time, enzyme supplies

were limited, many concepts of the structure and function of lipases were largely

speculative, and their molecular genetics was unknown. Since then, substantial ef-

forts in numerous laboratories, including the work described here, have built the

82 4 Cloning, Mutagenesis, and Biochemical Properties