Interfacial Activation of Triglyceride Lipase from Thermomyces (Humicola) lanuginosa: Kinetic Parameters and a Basis for Control of the Lid

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• 作者：Otto G. Berg ; Yolanda Cajal ; Glenn L. Butterfoss ; Ronald L. Grey ; M. Asuncion Alsina ; Bao-Zhu Yu ; and Mahendra K. Jain
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：May 12, 1998
• 年：1998
• 卷：37
• 期：19
• 页码：6615 - 6627
• 全文大小：199K
• 年卷期：v.37,no.19(May 12, 1998)
• ISSN：1520-4995

文摘

A strategy is developed to analyze steady-statekinetics for the hydrolysis of a soluble substratepartitioned into the interface by an enzyme at the interface. Thefeasibility of this approach to obtaininterfacial primary kinetic and equilibrium parameters is demonstratedfor a triglyceride lipase. Analysisfor phospholipase A₂ catalyzed hydrolysis of rapidlyexchanging micellar (Berg et al. (1997) Biochemistry36, 14512-14530) and nonexchangeable vesicular (Berg et al.,(1991) Biochemistry 30, 7283-7291)phospholipids is extended to include the case of a substrate that doesnot form the interface. The triglyceridelipase (tlTGL) from Thermomyces (formerly Humicola)lanuginosa hydrolyzes p-nitrophenylbutyrateortributyrin partitioned in the interface of1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG)vesiclesat a rate that is more than 100-fold higher than that for themonodispersed substrate or for the substratepartitioned into zwitterionic vesicles. Catalysis and activationis not seen with the S146A mutant withoutthe catalytic serine-146; however, it binds to the POPG interface withthe same affinity as the WT. ThusPOPG acts as a diluent surface to which the lipase binds in an active,or "open", form for the catalyticturnover; however, the diluent molecules have poor affinity for theactive site. Analysis of the substrateand the diluent concentration dependence of the rate of hydrolysisprovides a basis for the determinationof the primary interfacial catalytic parameters. As a competitivesubstrate, tributyrin provided a checkfor the apparent affinity parameters. Nonidealities from thefractional difference in the molecular areasin interfaces are expressed as the area correction factor and can beinterpreted as a first-order approximationfor the interfacial activity coefficient. The basis for theinterfacial activation of tlTGL on anionic interfaceis attributed to cationic R81, R84, and K98 in the "hinge" aroundthe 86-93 "lid" segment of tlTGL.