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Interaction of Lipoprotein Lipase With P-Nitrophenyl N-alkylcarbamates: Kinetics, Mechanism, and Analogy to the Acylenzyme Mechanism

Interaction of Lipoprotein Lipase With P-Nitrophenyl N-alkylcarbamates: Kinetics, Mechanism, and Analogy to the Acylenzyme Mechanism

H C Shin, D M Quinn

Biochemistry. 1992 Jan 28;31(3):811-8.

PMID: 1731939

Abstract:

The interaction of lipoprotein lipase with p-nitrophenyl N-alkylcarbamates [PNPOC(=O)-NHCnH2n+1; n = 4, 8, and 12] proceeds by the three-stage mechanism shown below. After reversible [formula: see text] formation of the enzyme-carbamate complex (EC), rapid carbamylation (kc) precedes slow decarbamylation. Therefore, in short-term assays (less than or equal to 30 min) of lipoprotein lipase catalyzed hydrolysis of p-nitrophenyl butyrate, activity is rapidly lost. The inhibition by p-nitrophenyl N-butylcarbamate follows saturation kinetics, which allows determination of Kc = 5.4 +/- 0.9 microM and kc = (4.9 +/- 0.7) x 10(-2)s-1. Saturation kinetics are not observed for the longer inhibitors p-nitrophenyl N-octylcarbamate and p-nitrophenyl N-dodecylcarbamate. Rather, plots of the pseudo-first-order rate constant for activity loss versus inhibitor concentration are concave upward, consistent with inhibitor binding to two sites on the enzyme. The inhibition phase is sufficiently rapid that p-nitrophenyl N-octylcarbamate can be used to titrate enzyme active sites. On the other hand, long-term assays (greater than 5 h) show sequential inhibition and activity return phases, and from the activity return phase kd is calculated. The long-term activity time course is accurately simulated by Runge-Kutta integration of the differential equations for the three-stage mechanism. These approaches have been used to characterize the kinetics of interaction of the enzyme with the carbamate inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP59837148	p-Nitrophenyl galacto-N-bioside		59837-14-8	Price

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