Acyl-chain specificity of human milk bile-salt-activated lipase.
In order to probe the active-site structure of human milk bile-salt-activated lipase (BAL), the kinetics of the BAL-catalysed reaction were studied using monoesters as substrates. Among the fatty acyl chains, ranging from C8 to C16 of monoacylglycerols in a single equimolar assay mixture, there was a consistent trend of increased reactivity with decreased fatty-acyl-chain length for both the basal and taurocholate-stimulated activities of BAL. In addition, the detection of hydrolysis of long-chain monoacylglycerols in the absence of bile salt indicates that it is possible for the long-chain fatty acid monoester to form an enzyme-substrate complex with the basal form of BAL. I further examined the reaction kinetics of BAL with water-soluble short-chain esters of p-nitrophenol. The results indicated that there is a consistent trend towards a decreased Michaelis-Menten constant with increased acyl-chain length. Therefore it was concluded that the decreased reactivity with increased acyl-chain length of acylglycerols is probably not a consequence of the lowered affinity of the substrate for the enzyme. The fact that butyrate ester has the optimum acyl chain to be a substrate of BAL can be attributed to its acyl-chain length being long enough for interaction with the active centre of BAL and short enough to provide adequate positioning of the ester bond for transition state complex formation. The calculated free energy of BAL catalysis based on the derived kinetic parameters provides additional insight into the effect on the enzyme-substrate interaction of increasing the number of methylene groups in the acyl chain of substrates.
Protein Studies Program, Oklahoma Medical Research Foundation.
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Wang
D001665:Binding Sites; D002384:Catalysis; D005989:Glycerides / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008049:Lipase / Q000378:metabolism*; D008066:Lipolysis; D002787:Sterol Esterase; D013656:Taurocholic Acid / Q000378:metabolism
D005989:Glycerides; D013656:Taurocholic Acid; C054784:bile salt-stimulated lipase; D002787:Sterol Esterase; D008049:Lipase