Kinetics of N-glutaryl-L-phenylalanine p-nitroanilide hydrolysis catalyzed by alpha-chymotrypsin in aqueous solutions of dodecyltrimethylammonium bromide.
The rate of hydrolysis of N-glutaryl-L-phenylalanine p-nitroanilide (GPNA) catalyzed by alpha-chymotrypsin (alpha-CT) has been measured in aqueous solutions of dodecyltrimethylammonium bromide (DTAB) at concentrations below and above the critical micelle concentration, as well as in the absence of surfactant. Under all the conditions employed, the reaction follows a Michaelis-Menten mechanism. The presence of the surfactant leads to superactivity below and above the critical micelle concentration (CMC), with a maximum reaction rate taking place near the CMC when the results are treated in terms of the analytical concentration of the substrate. A similar behavior was observed by working with the enzyme partially deactivated in the presence of 4 M urea. After correction to take into account the partitioning of the substrate between the micelles and the external media, the activity of the enzyme tends to remain almost constant above the corresponding CMCs. This results from a compensation of a decrease in the catalytic constant (k(cat)) and a decrease in the Michaelis constant (K(M)). The behavior of alpha-CT in the hydrolysis of GPNA in DTAB solutions is at variance with that previously reported for the hydrolysis of 2-naphthyl acetate in solutions of the same surfactant (E. Abuin, E. Lissi, R. Duarte, Langmuir 19 (2003) 5374). An explanation of the different effects of the surfactant on the behavior of the enzyme with both substrates is advanced, taking into account the complexity of the mechanism of the alpha-CT-mediated reaction, more specifically, in terms of different rate-limiting steps for the formation of the measured products.
Abuin
Elsa
E
Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 40, Correo 33, Santiago, Chile. eabuin@lauca.usach.cl
Lissi
Eduardo
E
Duarte
Roxanna
R
D002384:Catalysis; D002918:Chymotrypsin / Q000737:chemistry*; D004151:Dipeptides / Q000138:chemical synthesis* / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D012996:Solutions / Q000737:chemistry; D014867:Water / Q000737:chemistry
D004151:Dipeptides; D000644:Quaternary Ammonium Compounds; D012996:Solutions; D014867:Water; C007866:glutarylphenylalanine-4-nitroanilide; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; C022806:dodecyltrimethylammonium