Interfacial activation of triglyceride lipase from Thermomyces (Humicola) lanuginosa: Kinetic parameters and a basis for control of the lid

Otto G. Berg, Yolanda Cajal, Glenn Butterfoss, Ronald L. Grey, M. Asuncion Alsina, Bao Zhu Yu, Mahendra K. Jain

Research output: Contribution to journalArticle

Abstract

A strategy is developed to analyze steady-state kinetics for the hydrolysis of a soluble substrate partitioned into the interface by an enzyme at the interface. The feasibility of this approach to obtain interfacial primary kinetic and equilibrium parameters is demonstrated for a triglyceride lipase. Analysis for phospholipase A2 catalyzed hydrolysis of rapidly exchanging micellar (Berg et al. (1997) Biochemistry 36, 14512-14530) and nonexchangeable vesicular (Berg et al., (1991) Biochemistry 30, 7283-7291) phospholipids is extended to include the case of a substrate that does not form the interface. The triglyceride lipase (tlTGL) from Thermomyces (formerly Humicola) lanuginosa hydrolyzes p-nitrophenylbutyrate or tributyrin partitioned in the interface of 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphoglycerol (POPG) vesicles at a rate that is more than 100-fold higher than that for the monodispersed substrate or for the substrate partitioned into zwitterionic vesicles. Catalysis and activation is not seen with the S 146A mutant without the catalytic serine-146; however, it binds to the POPG interface with the same affinity as the WT. Thus POPG acts as a diluent surface to which the lipase binds in an active, or 'open', form for the catalytic turnover; however, the diluent molecules have poor affinity for the active site. Analysis of the substrate and the diluent concentration dependence of the rate of hydrolysis provides a basis for the determination of the primary interfacial catalytic parameters. As a competitive substrate, tributyrin provided a check for the apparent affinity parameters. Nonidealities from the fractional difference in the molecular areas in interfaces are expressed as the area correction factor and can be interpreted as a first-order approximation for the interfacial activity coefficient. The basis for the interfacial activation of tlTGL on anionic interface is attributed to cationic R81, R84, and K98 in the 'hinge' around the 86-93 'lid' segment of tlTGL.

Original languageEnglish (US)
Pages (from-to)6615-6627
Number of pages13
JournalBiochemistry
Volume37
Issue number19
DOIs
StatePublished - May 12 1998

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Lipase
Kinetic parameters
Hydrolysis
Chemical activation
Biochemistry
Substrates
Phospholipases A2
Catalysis
Serine
Catalytic Domain
Phospholipids
Kinetics
Activity coefficients
Enzymes
Hinges
Molecules
tributyrin

ASJC Scopus subject areas

  • Biochemistry

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Interfacial activation of triglyceride lipase from Thermomyces (Humicola) lanuginosa : Kinetic parameters and a basis for control of the lid. / Berg, Otto G.; Cajal, Yolanda; Butterfoss, Glenn; Grey, Ronald L.; Alsina, M. Asuncion; Yu, Bao Zhu; Jain, Mahendra K.

In: Biochemistry, Vol. 37, No. 19, 12.05.1998, p. 6615-6627.

Research output: Contribution to journalArticle

Berg, Otto G. ; Cajal, Yolanda ; Butterfoss, Glenn ; Grey, Ronald L. ; Alsina, M. Asuncion ; Yu, Bao Zhu ; Jain, Mahendra K. / Interfacial activation of triglyceride lipase from Thermomyces (Humicola) lanuginosa : Kinetic parameters and a basis for control of the lid. In: Biochemistry. 1998 ; Vol. 37, No. 19. pp. 6615-6627.
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T2 - Kinetic parameters and a basis for control of the lid

AU - Berg, Otto G.

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AU - Butterfoss, Glenn

AU - Grey, Ronald L.

AU - Alsina, M. Asuncion

AU - Yu, Bao Zhu

AU - Jain, Mahendra K.

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N2 - A strategy is developed to analyze steady-state kinetics for the hydrolysis of a soluble substrate partitioned into the interface by an enzyme at the interface. The feasibility of this approach to obtain interfacial primary kinetic and equilibrium parameters is demonstrated for a triglyceride lipase. Analysis for phospholipase A2 catalyzed hydrolysis of rapidly exchanging micellar (Berg et al. (1997) Biochemistry 36, 14512-14530) and nonexchangeable vesicular (Berg et al., (1991) Biochemistry 30, 7283-7291) phospholipids is extended to include the case of a substrate that does not form the interface. The triglyceride lipase (tlTGL) from Thermomyces (formerly Humicola) lanuginosa hydrolyzes p-nitrophenylbutyrate or tributyrin partitioned in the interface of 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphoglycerol (POPG) vesicles at a rate that is more than 100-fold higher than that for the monodispersed substrate or for the substrate partitioned into zwitterionic vesicles. Catalysis and activation is not seen with the S 146A mutant without the catalytic serine-146; however, it binds to the POPG interface with the same affinity as the WT. Thus POPG acts as a diluent surface to which the lipase binds in an active, or 'open', form for the catalytic turnover; however, the diluent molecules have poor affinity for the active site. Analysis of the substrate and the diluent concentration dependence of the rate of hydrolysis provides a basis for the determination of the primary interfacial catalytic parameters. As a competitive substrate, tributyrin provided a check for the apparent affinity parameters. Nonidealities from the fractional difference in the molecular areas in interfaces are expressed as the area correction factor and can be interpreted as a first-order approximation for the interfacial activity coefficient. The basis for the interfacial activation of tlTGL on anionic interface is attributed to cationic R81, R84, and K98 in the 'hinge' around the 86-93 'lid' segment of tlTGL.

AB - A strategy is developed to analyze steady-state kinetics for the hydrolysis of a soluble substrate partitioned into the interface by an enzyme at the interface. The feasibility of this approach to obtain interfacial primary kinetic and equilibrium parameters is demonstrated for a triglyceride lipase. Analysis for phospholipase A2 catalyzed hydrolysis of rapidly exchanging micellar (Berg et al. (1997) Biochemistry 36, 14512-14530) and nonexchangeable vesicular (Berg et al., (1991) Biochemistry 30, 7283-7291) phospholipids is extended to include the case of a substrate that does not form the interface. The triglyceride lipase (tlTGL) from Thermomyces (formerly Humicola) lanuginosa hydrolyzes p-nitrophenylbutyrate or tributyrin partitioned in the interface of 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphoglycerol (POPG) vesicles at a rate that is more than 100-fold higher than that for the monodispersed substrate or for the substrate partitioned into zwitterionic vesicles. Catalysis and activation is not seen with the S 146A mutant without the catalytic serine-146; however, it binds to the POPG interface with the same affinity as the WT. Thus POPG acts as a diluent surface to which the lipase binds in an active, or 'open', form for the catalytic turnover; however, the diluent molecules have poor affinity for the active site. Analysis of the substrate and the diluent concentration dependence of the rate of hydrolysis provides a basis for the determination of the primary interfacial catalytic parameters. As a competitive substrate, tributyrin provided a check for the apparent affinity parameters. Nonidealities from the fractional difference in the molecular areas in interfaces are expressed as the area correction factor and can be interpreted as a first-order approximation for the interfacial activity coefficient. The basis for the interfacial activation of tlTGL on anionic interface is attributed to cationic R81, R84, and K98 in the 'hinge' around the 86-93 'lid' segment of tlTGL.

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