Lipase-catalysed hydrolysis of short-chain substrates in solution and in emulsion: a kinetic study.
We have studied the enzymatic hydrolysis of solutions and emulsions of vinyl propionate, vinyl butyrate and tripropionin by lipases of various origin and specificity. Kinetic studies of the hydrolysis of short-chain substrates by microbial triacylglycerol lipases from Rhizopus oryzae, Mucor miehei, Candida rugosa, Candida antarctica A and by (phospho)lipase from guinea-pig pancreas show that these lipolytic enzymes follow the Michaelis-Menten model. Surprisingly, the activity against solutions of tripropionin and vinyl esters ranges from 70% to 90% of that determined against emulsions. In contrast, a non-hyperbolic (sigmoidal) dependence of enzyme activity on ester concentration is found with human pancreatic lipase, triacylglycerol lipase from Humicola lanuginosa (Thermomyces lanuginosa) and partial acylglycerol lipase from Penicillium camembertii and the same substrates. In all cases, no abrupt jump in activity (interfacial activation) is observed at substrate concentration corresponding to the solubility limit of the esters. Maximal lipolytic activity is always obtained in the presence of emulsified ester. Despite progress in the understanding of structure-function of lipases, interpretation of the mode of action of lipases active against solutions of short-chain substrates remains difficult. Actually, it is not known whether these enzymes, which possess a lid structure, are in open or/and closed conformation in the bulk phase and whether the opening of the lid that gives access to the catalytic triad is triggered by interaction of the enzyme molecule with monomeric substrates or/and multimolecular aggregates (micelles) both present in the bulk phase. From the comparison of the behaviour of lipases used in this study which, in some cases, follow the Michaelis-Menten model and, in others, deviate from classical kinetics, it appears that the activity of classical lipases against soluble short-chain vinyl esters and tripropionin depends not only on specific interaction with single substrate molecules at the catalytic site of the enzyme but also on physico-chemical parameters related to the state of association of the substrate dispersed in the aqueous phase. It is assumed that the interaction of lipase with soluble multimolecular aggregates of tripropionin or short-chain vinyl esters or the formation of enzyme-substrate mixed micelles with ester bound to lipase, might represent a crucial step that triggers the structural transition to the open enzyme conformation by displacement of the lid.
Nini
L
L
Laboratory of Applied Biochemistry, Faculty of Sciences and Technology, St-Jérôme. Marseille, France.
Sarda
L
L
Comeau
L C
LC
Boitard
E
E
Dubès
J P
JP
Chahinian
H
H
D000818:Animals; D002087:Butyrates / Q000378:metabolism; D002175:Candida; D004655:Emulsions; D006168:Guinea Pigs; D006868:Hydrolysis; D007700:Kinetics; D008049:Lipase / Q000378:metabolism / Q000494:pharmacology*; D011422:Propionates / Q000378:metabolism; D012233:Rhizopus; D012996:Solutions; D014280:Triglycerides / Q000378:metabolism; D014753:Vinyl Compounds / Q000378:metabolism
D002087:Butyrates; D004655:Emulsions; D011422:Propionates; D012996:Solutions; D014280:Triglycerides; D014753:Vinyl Compounds; C095498:vinyl butyrate; D008049:Lipase; C075987:pancreatic lipase-related protein 1; C031627:tripropionin