Competitive inhibition by substrates of the esterification reaction between l-phenylalanine and d-glucose catalysed by the lipases of Rhizomucor miehei and Candida rugosa

Abstract

A kinetic study on esterification between d-glucose and l-phenylalanine catalysed by lipases from Rhizomucor miehei (RML) and Candida rugosa (CRL) in organic media investigated in detail showed that both the lipases followed a Ping-Pong Bi-Bi mechanism with two distinct types of competitive inhibitions. Graphical double reciprocal plots and computer simulation studies showed that competitive double substrate inhibition took place at higher concentrations leading to dead-end inhibition in the case of RML and in the case of CRL, inhibition only by d-glucose at higher concentrations leading to dead-end lipase–d-glucose complexes. An attempt to obtain the best fit of these kinetic models through curve-fitting yielded in good approximation, the apparent values of important kinetic parameters, RML: kcat = 2.24 ± 0.23 mM h−1 (mg protein)−1, Km l-phenylalanine = 95.6 ± 9.7 mM, Km d-glucose = 80.0 ± 8.5 mM, Ki l-phenylalanine = 90.0 ± 9.2 mM, Ki d-glucose = 13.6 ± 1.42 mM; CRL: kcat = 0.51 ± 0.06 mM h−1 (mg protein)−1, Km l-phenylalanine = 10.0 ± 0.98 mM, Km d-glucose = 6.0 ± 0.64 mM, Ki d-glucose = 8.5 ± 0.81 mM.