Comparative kinetic study of lipases A and B from Candida rugosa in the hydrolysis of lipid p-nitrophenyl esters in mixed micelles with Triton X-100.
(1) Lipases A and B from Candida rugosa catalyzing the hydrolysis of esters in micellar media have been characterized kinetically by studies on substrate specificity, rate equation forms and modeling of enzyme mechanisms. (2) The study on specificity revealed that both lipases are non-specific esterases with similar activity against lipid p-nitrophenyl esters micellized with Triton X-100. The slight difference was that lipase A has its maximum activity centered in the caprylate while that of lipase B is in the laurate. (3) Kinetic studies for both lipases were carried out with p-nitrophenyl laurate under three experimental conditions: (I) the molar fraction of substrate is fixed and the bulk concentration of substrate and Triton X-100 are varied; (II) the bulk concentration of substrate is held constant and the molar fraction of substrate and bulk concentration of Triton X-100 are varied; and (III) the bulk concentration of Triton X-100 is held constant but the bulk concentration of substrate and molar fraction of substrate are varied. (4) In case I, a similar Michaelis-Menten behaviour was observed with both lipases; the curve fitting gave kappcat/Kappm values of 3.0.10(5) and 5.6.10(5) s-1 M-1 for lipases A and B respectively. In case II, for both lipases the relationship between rate and the molar fraction of substrate required a fitting equation of 2:2 degree polynomial quotient. In case III, both lipases showed non-Michaelian behaviour with concave-up curves in the Eadie-Hofstee plot, a minimum degree of 2:2 in substrate concentration being detected for the rate equation. (5) The above results are interpreted in terms of the hypothesis that the mechanism of both lipases must include at least two different inputs for the molecule of substrate which would explain the quadratic terms observed in the rate equation.
Redondo
O
O
Departamento de Química Física, Facultad de Farmacia, Universidad de Salamanca, Spain.
Herrero
A
A
Bello
J F
JF
Roig
M G
MG
Calvo
M V
MV
Plou
F J
FJ
Burguillo
F J
FJ
D002175:Candida / Q000201:enzymology*; D002210:Caprylates; D004952:Esters / Q000378:metabolism*; D006868:Hydrolysis; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D007850:Lauric Acids; D008049:Lipase / Q000378:metabolism*; D008823:Micelles; D008956:Models, Chemical; D009596:Nitrophenols; D013379:Substrate Specificity
D002210:Caprylates; D004952:Esters; D007527:Isoenzymes; D007850:Lauric Acids; D008823:Micelles; D009596:Nitrophenols; C030358:lauric acid; D008049:Lipase; C031492:octanoic acid; C024836:4-nitrophenol