Inhibition kinetics of camel lens zeta-crystallin: multiple inhibition studies

The inhibition of camel lens zeta-crystallin by nitrofurantoin (NF) was uncompetitive with respect to co-factor NADPH, (Ki = 90 microM) and competitive with respect to the substrate 9,10-phenanthrenequinone (PQ), (Ki = 50 microM). Inhibition at micromolar concentrations was also observed with dicoumarol, NADP+ and cibacron blue (CB). Theorell-Yonetani double-inhibition analysis showed that NF and dicoumarol were mutually exclusive inhibitors against PQ. However, analysis of NF and NADP+ by a double-inhibition plot showed that they simultaneously bind to the enzyme molecule. These studies demonstrate that NF and dicoumarol share the same site so that both molecules are prevented from binding at the same time, while NF and NADP+ can bind simultaneously to different sites on the enzyme. Although CB was noncompetitive with respect to PQ, double inhibition analysis showed that CB and dicoumarol or NF were mutually exclusive inhibitors against PQ, implying a distinct mode of inhibition for CB.