Abstract
Pancreatic carboxylester lipase catalyzes the exchange of 18O between water and 13,16-cis,cis-doco-sadienoic acid (DA) in monolayers at the argon-buffer interface (Muderhwa, J.M., Schmid, P.C., and Brockman, H.L. (1992) Biochemistry 31, 141). In mixed monolayers of 18O, 18O-DA and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), both the extent and mechanism of 18O exchange show characteristics of a critical transition in the range of 0.5-0.6 mol fraction of DA (Muderhwa, J.M., and Brockman, H. L. (1992) Biochemistry 31, 149). To determine if the regulatory behavior exhibited on this type of surface is limited to members of the carboxylester lipase gene family (cholinesterases), comparable experiments were performed with a genetically and functionally unrelated lipase, pancreatic colipase-dependent lipase (PL). PL readily catalyzed the exchange of 18O between water and the carboxyl group of DA with enzyme at either monolayer or catalytic levels in the fatty acid-buffer interface. The oxygen exchange reaction obeyed a random, sequential mechanism, indicating that the dissociation of the enzyme.DA complex is much faster than the rate-limiting step in the overall exchange process. Kinetic analysis of oxygen exchange in pure DA monolayers showed a first-order dependence on interfacial PL and DA concentrations from which kcat/Km values were calculated. The oxygen exchange reaction proceeded with a rate constant of 16 x 10(-2) cm2 pmol-1 s-1, a value comparable to that for hydrolysis of the ester substrate, 1,3-dioleoylglycerol. With a monolayer of PL adsorbed to the interfacial phase, kcat/Km for oxygen exchange was about 600-fold lower than the value obtained with catalytic levels of adsorbed enzyme, indicating a possible restriction of substrate diffusion in the protein-covered fatty acid monolayer. With constant bulk PL concentration and mixed lipid monolayers containing DA and the non-substrate lipid, POPC, the extent of oxygen exchange increased abruptly as the abundance of DA in the interface was increased from 0.5 to 0.6 mol fraction. Concomitant with this critical transition was a change in the apparent mechanism of oxygen exchange from coupled to random, sequential. For both the extent of oxygen exchange and its mechanism shift, the critical transition was independent of the lipid packing density, i.e. surface pressure, of the interface. These results show that PL responds similarly to carboxylester lipase with respect to changes in interfacial lipid mole fraction in DA-POPC surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
Pancreaticcarboxylesterlipasecatalyzestheexrespect to changes in interfacial lipidmole fraction in change of ”0 between water and 13,16-cis,cis-doco- DA-POPC surfaces
This research has shown that theoverall water and the carboxyl group ofDA with enzyme at catalytic process involves twodistinct steps: adsorption of the either monolayer or catalytic levels in the fatty acid- enzyme from the bulk aqueous phase to thesurface phase and buffer interface
The oxygen exchange reaction proceeded with a rate constant of 16 x cm2 pmol-1 s-’, a value comparable to that for hydrolysis of theestersubstrate, 1,3-dioleoylglycerol
Summary
Pancreaticcarboxylesterlipasecatalyzestheexrespect to changes in interfacial lipidmole fraction in change of ”0 between water and 13,16-cis,cis-doco- DA-POPC surfaces. With constant bulkPL concentration andmixed scopic lateral phase separation [7,8] In model systems, such lipid monolayers containing DA and the non-substrate changes are known to regulate the activities of (phoslipid, POPC, the extentof oxygen exchange increased pho)lipases themselves (9,lO) and other membrane-mediated abruptly as the abundance of DA in the interface was cellular functions, such as calcium transport (ll),cell fusion increased from 0.5 to 0.6 mol fraction. A critical transitionwas observed resulting solution were assayedforactiveenzyme content usinga fortheextent of CEL-catalyzedexchange of l8O between pH-stat (Radiometer) assaydescribedpreviously [41, 42] with one water and the carboxyl group of 13,16-cis,cis-docosadienoic acid (DA) in mixed monolayers with POPC [26] With both ester and fattaycid substrates, the critical transitioonccurred modification.Because PL adsorption was low whenmeasured at dilute bulk PL concentrations (0.01-5 nM), the assay mixture consisted of 2 mlof an emulsion of 126 mM tributyrin,preparedas reported elsewhere [28], containing units of colipase. Hypothesis, we have studied the abilityof PL tocatalyze the hydrolysis of 1,3-dioleoylglycerol (1,3-DO) and the exchange of ” 0 between water and fattyacid in monolayer films with
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