Cysteine residues in human lysosomal acid lipase are involved in selective cholesteryl esterase activity.
Human lysosomal acid lipase (LAL) catalyses the deacylation of triacylglycerol and cholesteryl esters in the acidic lysosomal compartment. Treatment of LAL with the reducing agent dithiothreitol affected the triacylglycerol and cholesteryl esterase activities differentially, suggesting the involvement of cysteine residues in determining substrate specificity. To identify the residues involved, human LAL cDNA, under the control of the T7 promoter and tagged with a herpes simplex virus coding epitope, was specifically mutated in order to introduce single amino acid substitutions of each of the six cysteine residues of mature LAL. All Cys-227 mutants showed selectively decreased activity towards cholesteryl oleate, while preserving that towards trioleylglycerol. Substitutions of Cys-236, Cys-240 and Cys-244 affected catalysis towards the two substrates to a variable degree, depending on the side chain of the amino acid introduced. The replacement of Cys-41 or Cys-188 did not result in the preferential cleavage of either one of the two substrates. These data indicate that Cys-227, Cys-236, Cys-240 and Cys-244 play a crucial role in determining LAL substrate specificity. We propose that these cysteine residues are involved in the hydrolysis of cholesteryl ester by affecting selectively the access of this substrate to the catalytic active site. In addition, the fact that the catalytic activity is never completely abolished in cysteine mutants demonstrates that LAL is not a thiol enzyme.
Pagani
F
F
International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
Pariyarath
R
R
Stuani
C
C
Garcia
R
R
Baralle
F E
FE
D003545:Cysteine / Q000235:genetics / Q000502:physiology*; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D008049:Lipase / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D008247:Lysosomes / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D019163:Reducing Agents / Q000494:pharmacology; D002787:Sterol Esterase / Q000378:metabolism*; D013379:Substrate Specificity / Q000187:drug effects / Q000235:genetics
D019163:Reducing Agents; D002787:Sterol Esterase; D008049:Lipase; D003545:Cysteine; D004229:Dithiothreitol