Mutational analysis of the "regulatory module" of hormone-sensitive lipase.
Hormone-sensitive lipase (HSL) is a rate-limiting enzyme in lipolysis that displays broad substrate specificity. HSL function is regulated by reversible phosphorylation that occurs within a 150 aa "regulatory module" of the protein. The current studies used mutational analysis to dissect the contribution of the "regulatory module" in HSL activity and substrate specificity. Deletion of the entire "regulatory module" or replacement of the "regulatory module" with the "lid" of lipoprotein lipase resulted in enzymatically inactive proteins. Deletion of sequentially longer stretches of the "regulatory module" resulted in a stepwise reduction in hydrolytic activity. Analysis of 7-19 amino acid deletional mutants that spanned the "regulatory module" showed that the N-terminal partial deletion mutants retained normal hydrolytic activity and activation by PKA. In contrast, the C-terminal partial deletion mutants displayed reduced hydrolytic activities, with preferential loss of activity against lipid-, as opposed to water-soluble, substrates. Single amino acid mutations of F650C, P651A, and F654D reduced activity against lipid-, but not water-soluble, substrates. The current results suggest that the length of the "regulatory module" and specific sequences within the C-terminal portion of the "regulatory module" of HSL (amino acids 644-683) are crucial for activity and appear to be responsible for determining lipase activity.
Wang
Jining
J
VA Palo Alto Health Care System, Palo Alto, California, USA.
Shen
Wen-Jun
WJ
Patel
Shailja
S
Harada
Kenji
K
Kraemer
Fredric B
FB
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis / Q000379:methods; D006868:Hydrolysis; D008055:Lipids / Q000737:chemistry; D008071:Lipoprotein Lipase / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D017384:Sequence Deletion; D002787:Sterol Esterase / Q000737:chemistry* / Q000235:genetics*; D013696:Temperature; D014162:Transfection
D008055:Lipids; D011993:Recombinant Fusion Proteins; D002787:Sterol Esterase; D008071:Lipoprotein Lipase