The histidines reacting with ethoxyformic anhydride in porcine pancreatic lipase: their relationships with enzyme activity.
The activities of porcine pancreatic lipase (449 amino acid residues) toward two different substrates, p-nitrophenylacetate and tributyrylglycerol, and their dependence on histidine ethoxyformylation were studied. In parallel, the ethoxyformylation of the lipase fragment constituting the C-terminal sequence of lipase (residues 336 to 449) was also investigated. This fragment was found to have retained the ability of lipase to catalyse p-nitrophenylacetate hydrolysis. The first histidine to react either in lipase or in the lipase fragment was His-354. The activities of the two compounds toward p-nitrophenyl-acetate were lost but that of the enzyme toward tributyrylglycerol was almost entirely retained. When a larger excess of ethoxyformic anhydride was used for the lipase reaction, 2.8 histidine residues were ethoxyformylated and characterised as His-354, His-156 and His-75, which resulted in an 85% inhibition of the tributyrylglycerol hydrolysis by the enzyme. Hydroxylamine treatment reactivated most of the lipase and lipase fragment. This is the first demonstration that the two lipase activities are not associated with the same active site. The loss of activity toward triacylglycerol hydrolysis suggests that His-156 and/or His-75 belong(s) to the active site or that a conformational change resulting from the ethoxyformylation renders the lipase inactive.
De Caro
J D
JD
Centre de Biochimie et de Biologie Moléculaire du CNRS, Marseille, France.
Guidoni
A A
AA
Bonicel
J J
JJ
Rovery
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004047:Diethyl Pyrocarbonate / Q000378:metabolism*; D005561:Formates / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D008049:Lipase / Q000378:metabolism*; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000378:metabolism; D010179:Pancreas / Q000201:enzymology*; D013379:Substrate Specificity; D013552:Swine; D014280:Triglycerides / Q000378:metabolism
D005561:Formates; D009596:Nitrophenols; D014280:Triglycerides; D006639:Histidine; C008642:4-nitrophenyl acetate; D008049:Lipase; D004047:Diethyl Pyrocarbonate; C005830:tributyrin