Hi there,
We are working on a new compound for a enzymatic bi substrate reaction and our goal is that find the mechanism of action of the compound.
Does anyone know how the mechanism of action of inhibitor in enzymatic bi substrate reactions should be determined? In some papers that I have seen, they have used single substrate - Michaelis Menten model for bi substrate reactions that does not make sense for me. Do you think methods like double reciprocal plots work for finding mechanism of action of inhibitors in enzymatic reactions that involve two substrates? What is pros and cons of these methods
Special thanks in advance for your kind reply
Before undertaking a complex investigation of the inhibitor, do everything you can to exclude the possibility that it behaves in a non-specific manner. For example, compounds can inhibit because they form soluble aggregates, contain contaminants, or act as denaturants. Use the purest sample of the inhibitor you can get. Measure its solubility in the assay buffer. Do dynamic light scattering, if it is available, to check for aggregation. Test the effect on the potency of the inhibitor of increasing the enzyme concentration and of including 0.01% Triton X-100 in the assay (these last 3 tests were proposed by Brian Shoichet). Measure the Hill slope of an IC50 curve (in most cases, it should be 1).
Remember that to do steady-state kinetics with the inhibitor, the inhibitor concentrations, like the substrate concentrations, must be much greater than the enzyme concentration.
Steady-state kinetics can be used to elucidate the kinetic mode of inhibition of bisubstrate enzymes, particularly for competitive inhibitors. Traditionally, reciprocal plots and replots were used. The methodology is very thoroughly described in Segel's book "Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems."
Under the right circumstances, you can take the approach of saturating one of the substrates and varying the other substrate and the inhibitor concentrations in order to reduce the system to a pseudo-single substrate one. Before doing this, you should find out whether there is substrate inhibition by either of the substrates, which has to be taken into account.
Another way to go about the investigation takes advantage of the availability of software for nonlinear regression. A 3-D matrix of initial rate data is collected in which both substrates and the inhibitor concentration are varied. A set of initial rate equations that might correspond to the kinetic mechanism is tested for goodness of fit (chi-squared) to the data. This procedure can show which models are the best fit. It is very helpful when doing this if you already know the kinetic rate equation for the uninhibited reaction.
Steady-state kinetics is not the only way to investigate the mechanism. You can also look directly at inhibitor binding by a wide variety of biophysical approaches. These results can also help inform the kinetic investigation, and the results can be easier to interpret. To begin, you can ask how tightly the inhibitor binds to the enzyme in the absence of both substrates or in the presence of each of them. You can also ask how the presence of the inhibitor affects binding of each of the substrates. Ultimately, x-ray crystallography can show you exactly where the inhibitor binds.
You can learn more from Robert A. Copeland's book "Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide for Medicinal Chemists and Pharmacologists"
Determine Km and Vmax values for each of the substrates, and in a bisubstrate reaction it's quite likely that the enzyme will prefer that substrate first for whichit has a greater Vmax and lower Km.
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