The enzymes of the acid lipase family likely catalyze the hydrolysis of ester bonds of acylglycerols and cholesteryl esters via an acylenzyme mechanism,123 such as that utilized by enzymes of the α/β hydrolase family and outlined in Scheme 5. Chemical modification studies suggest that the enzymes are serine esterases, but loss of activity is also noted in the presence of sulfhydryl-modifying reagents. The serine esterase mechanism is supported by a comparative site-specific mutagenesis study of HLAL and HGL.123 Both HLAL and HGL contain two lipase consensus sequences -Gly-X-Ser-X- Gly- (X = variable amino acid), one centered on Ser99 and one on Ser153, that contain putative active site nucleophiles. Replacement of Ser153 in HLAL with Thr produced a mutant enzyme that had no activity against cholesteryl oleate, triolein, or tributyrin substrates. However, replacement of Ser99 by Thr gave a mutant HLAL that retained ∼30–70% of the activity of the wild-type enzyme. Parallel mutagenesis experiments in HGL showed that Ser153 was necessary for activity against triolein or tributyrin, but Ser99 was not. Therefore, Ser153 is implicated as the nucleophile in the catalytic triads of acid lipases.
Eight Asp residues that are conserved in the three acid lipases were replaced by Gly in a series of single mutants of HLAL.123 Three of the mutants (Asp89Gly, Asp124Gly, and Asp257Gly) retained significant catalytic activity toward both cholesteryl oleate and triolein substrates, and therefore cannot contribute the carboxylate residue to the catalytic triad. Three additional mutants (Asp93Gly, Asp130Gly, and Asp328Gly) retained significant activity toward triolein but were catalytically inactive toward cholesteryl oleate, establishing a differential role for these residues in the two lipid ester hydrolyses for which HLAL is a catalyst. However, substitution of either Asp324 or Asp331 by Gly abolished activity toward both substrates, and therefore one of these two residues is a likely component of the active site triad. This issue was settled by constructing a series of single mutants in which Asp324, Asp328, or Asp331 of HGL is replaced by Gly. Of these, the Asp328Gly and Asp331Gly mutants were almost as active as wild-type enzyme toward both triolein and tributyrin substrates, whereas the Asp324Gly mutant was devoid of activity. Therefore, comparative mutagenesis in HLAL and HGL suggests that Asp324 is the acid component of the catalytic triad.
Six His residues are conserved in HLAL, HGL, and RLL, and each of these was replaced by Gln in a series of single mutants.123 Substitutions of His262, His298, and His345 in HLAL gave mutant enzymes that retained significant catalytic activity, while replacements of the remaining three conserved residues His65, His274, and His353 gave enzymes that were catalytically inactive. Replacement of His274 in HGL by Gln gave a mutant enzyme that retained significant catalytic activity, and therefore either His65 or His353 was suggested to be the general acid/base element of the catalytic triad. Of these residues, His353 is favored because its sequence position gives an ordering of triad residues, that is, Ser153-Asp324-His353, that is like those found in numerous additional lipases and esterases.
Therefore, the acid lipases, like the esterases and lipases of the α/β hydrolase fold family and the peptidases of the serine protease families, have arrived by convergent evolution on the catalytic triad theme of transition state stabilization. However, the operation of active site triads in acid lipases raises a number of questions. In serine proteases and esterases, the pKa of the imidazole side chain of the active site His falls in the range 6–7.5.44,53 What structural features of the active sites of acid lipases lower the pKa of the His to such a degree that the enzymes have high activities at pH values as low as 2? Is His353 the general acid/base component of a traditional triad? Or does the protonated form of His353 serve solely as a general acid to assist the departure of the leaving group from the tetrahedral intermediate (cf. Schemes 1 and 5) or as an H-bond donor in the oxyanion hole? Do the acid lipases share topological features with esterases and lipases of the α/β hydrolase fold family, or do they possess a unique fold that defines a new supergene family? Answers to these and other questions must await the report of the first X-ray structure of an acid lipase.