Interaction of lipoprotein lipase with p-nitrophenyl N-alkylcarbamates: kinetics, mechanism, and analogy to the acylenzyme mechanism.
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)
Shin
H C
HC
Department of Chemistry, University of Iowa, Iowa City 52242.
Quinn
D M
DM
D000818:Animals; D001665:Binding Sites; D002219:Carbamates / Q000138:chemical synthesis / Q000494:pharmacology*; D002417:Cattle; D005260:Female; D007202:Indicators and Reagents; D007700:Kinetics; D008071:Lipoprotein Lipase / Q000378:metabolism*; D008433:Mathematics; D008892:Milk / Q000201:enzymology; D008962:Models, Theoretical; D009596:Nitrophenols / Q000494:pharmacology; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013997:Time Factors
D002219:Carbamates; D007202:Indicators and Reagents; D009596:Nitrophenols; D008071:Lipoprotein Lipase