CYSTEINE RESIDUES IN HUMAN LYSOSOMAL ACID LIPASE ARE INVOLVED IN SELECTIVE CHOLESTERYL ESTERASE-ACTIVITY

Human lysosomal acid lipase (LAL) catalyses the deacylation of triacyl glycerol and cholesteryl esters in the acidic lysosomal compartment. T reatment of LAL with the reducing agent dithiothreitol affected the tr iacylglycerol and cholesteryl esterase activities differentially, sugg esting the involvement of cysteine residues in determining substrate s pecificity. To identify the residues involved, human LAL cDNA, under t he control of the T7 promoter and tagged with a herpes simplex virus c oding epitope, was specifically mutated in order to introduce single a mino acid substitutions of each of the six cysteine residues of mature LAL. All Cys-227 mutants showed selectively decreased activity toward s cholesteryl oleate, while preserving that towards trioleylglycerol. Substitutions of Cys-236, Cys-240 and Cys-244 affected catalysis towar ds the two substrates to a variable degree, depending on the side chai n of the amino acid introduced. The replacement of Cys-41 or Cys-188 d id not result in the preferential cleavage of either one of the two su bstrates. These data indicate that Cys-227, Cys-236, Cys-240 and Cys-2 44 play a crucial role in determining LAL substrate specificity. We pr opose that these cysteine residues are involved in the hydrolysis of c holesteryl ester by affecting selectively the access of this substrate to the catalytic active site. In addition, the fact that the catalyti c activity is never completely abolished in cysteine mutants demonstra tes that LAL is not a thiol enzyme.