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

    Fingerprint

    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

    Cite this

    Berg, O. G., Cajal, Y., Butterfoss, G., Grey, R. L., Alsina, M. A., Yu, B. Z., & Jain, M. K. (1998). Interfacial activation of triglyceride lipase from Thermomyces (Humicola) lanuginosa: Kinetic parameters and a basis for control of the lid. Biochemistry, 37(19), 6615-6627. https://doi.org/10.1021/bi972998p

    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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