Enzyme kinetics of RNase present in testes

Abstract

Enzyme kinetics is one of the important parameter to be studied to know the inhibition studies of RNase A. RNase A is an allosteric enzyme which consists of an allosteric site in addition to the active site of the enzyme. With respect to allosteric enzymes two types of modulators namely positive and negative regulates the enzyme. Positive allosteric modulators increase the cooperitivity to other sites in positive manner for a multi subunit protein. So, K0.5 is usually decreased and Vmax found to be increased.1 Where as negative modulators decrease the affinity of substrate binding at one subunit of enzyme compared to other subunits of it. So, K0.5 found to be increased and Vmax decreases.1 O2 binding to the Hb is an example of positive cooperivity and feedback inhibition by allosteric enzyme is an example of negative cooperitivity. UV visible spectroscopic analysis in vitro proved RNase A is an allosteric enzyme and agarose gel electrophoresis analysis2 has proved that metosartan is an inhibitor of RNase A. Various plots of enzyme like Michaelis–Menton plot, Line weaverburk plot, Dixon plot and Eadiehofstee plot are used to know the K0.5 and Vmax of the enzyme in the presence and absence of drug. As RNase A is an allosteric enzyme it doesn’t follows Michaelsmenton kinetics as it consists of more than one active site. Allosteric enzymes give profound sigmoid curve with negative modulator and hyperbolic curve with positive modulator. Line weaver – Burk plot is not useful in case of allosteric enzymes to know whether the inhibition pattern is allosteric or not. Dixon plots are used to know the potency of inhibitor over the enzyme and Eadie – Hofstee plot is useful to know the Km/Vmax and Km. But it is difficult to know the Km and Vmax of the enzymes by EadieHofstee plot. Enzyme kinetics for each concentration is plotted and its effect of drug on enzyme was studied. The term Km is not used for allosteric enzymes instead K0.5 is used as the enzyme doesn’t follows Michaels menton kinetics. Properties of allosteric enzymes are as follows. Higher substrate concentration favours R state of the enzyme where as lower concentration favours T state where as other molecules like O2 and 2,3-Bis phosphor glycerate are regulators of allosteric enzymes.