My question refers to the fundamental problem of enzyme kinetics. I am working on a hydrolase which is specific for (lets say) substrate "A". I perform a real time kinetic assay to determine the Km, kat and Vmax for the given reaction. Hydrolysis of "A" gets completed within 30 min at given substrate (10 mM) and enzyme (10 nM) concentration. Then I an investigating the hydrolase activity of enzyme with analogues of "A". Some analogues (lets say) "B", "C" and "D" are showing positive activity. But in order to achieve the reaction (within instrument's detection limit) I increased the enzyme concentration by 100x at the same substrate (10 mM) concentration. "B" gets 80% converted into product in 60 min at same reaction condition. While "C" showed the 40% conversion in 4 hrs and "D" 20% in 6 hrs. I can say that my enzyme is active for those substrates but my question is how to calculate the kinetic parameters for those substrates. Does reporting the relative activity suffice and if yes how do I report? Is plotting MM kinetics is okay for such incomplete reactions? Or just reporting yes/no is suffice?
Hi there,
If you intention is to run Michaelis Menten equation then you actually have to determine initial velocities which is very different from the time needed to observe the completion of the reaction. Except if your system allows the use and the treatment of progression curve data (one kinetics experiment leading to Km and Vm determination, the hypothesis being there is no inhibition contribution of the product during the experiment).
So from your preliminary data, the enzyme has a preference for A then B, C and D. To be more accurate you have to go through Km, kcat determination (as long as E is always very low compared to substrate, it is OK).
Usually we first work on the affinity of an enzyme for a set of given substrates which may include natural or synthetic analogue, too. Then for the beast substrate try to determine the kinetic parameters in including Kcat, Vmax, Km etc. Otherwise there may be n number of substrates for the enzyme. What matters is the substrate for which enzyme has the maximum affinity, and the turn over (Kcat) number as there indicate efficiency of an enzyme.
You must determine Km and Vmax for each substrate using conditions in which you can measure initial reaction velocities reliably at various substrate concentrations. It may be necessary to use higher enzyme concentrations for substrates that react slowly, but you obviously have knowledge of enzyme concentration [E] so you can determine kcat for each substrate from Vmax = kcat[E]. A comparison of the efficiency of the enzyme acting on the different substrates is then possible from the individual kcat/Km ratios.
Peter
I must sadly say that the HPLC-photometric method uniquely gives true result (please see file; J Chrom B rat BIN LIP Km).
Surely, pure enzyme recognizes multiple substrates (please see file; Multiple hydrolase LIP). Further, we have recently found using affinity chromatographically that even pure avidin recognizes D,L-lipoic acid, D-biotin, Desthiobiotin, L-amino acids, and D-amino acids (expressed in the affinity-strength order) (please see files; lipoic acid avidin and D-Asp Avidin).
I must say again that the sensitive and reliable HPLC-photometric method can uniquely give the true answer or result.
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