remove the product somehow (for example another enzyme as mentioned above, or some other method, depends what the substance is), or play with the equilibrium. Is the reaction egzo or endoergic? If you start far enough from equilibrium, it has to be predominantly in one direction at least in the beginning.

Use different method for measuring the [substrate] and [product], IR for example? Would be easier to help if we knew the substrate and product.

Normally this sort of an assay uses a 2nd enzyme that removes one of the products.

My advice is to stay within the constraints of determining the initial velocity (i.e., when negligible product has formed to contribute to the reverse reaction) to measure these kinetics parameters, It is vastly more complex to extract kinetics parameters for reversible enzymes when the forward and reverse reactions are simultaneously occurring.

remove the product somehow (for example another enzyme as mentioned above, or some other method, depends what the substance is), or play with the equilibrium. Is the reaction egzo or endoergic? If you start far enough from equilibrium, it has to be predominantly in one direction at least in the beginning.

Use different method for measuring the [substrate] and [product], IR for example? Would be easier to help if we knew the substrate and product.

I would suggest a few approaches. First, you should only be looking at the initial rates to determine Km and kcat. If you use a very short time scale, there should be very little product (compared to substrate) and thus there should be little contribution to the absorbance from the product. The best way to do this would be on a stop flow or rapid quench system, where you can look at very short time scales. If this is not satisfactory, and you have the product available as well, you can do initial rates of substrate and initial rates of products. As long as there is some slight difference in the absorbance of the two, you can get two equations in two unknowns and figure out the rates for both reactions. If you are having trouble seeing any difference in substrate or product via UV, you can try getting spectra at different pH's. Sometimes, molecules that have similar spectra will have drastically different absorption spectra at high or low pH values. You may be able to run your assay, quench the reaction, change the pH and then measure the concentrations. If none of this works, you may need to add an enzyme that removes your product as the reaction proceeds. If your enzyme is in a pathway, the next enzyme in the pathway can be added to remove product as its produced. Hope this helps.

Try to do forward reaction with substrate in vast excess - so the product will have just slight impact on forward reaction, similarly do it for backward reaction.

I would also suggest high substrate concentrations if there is no substrate inhibition.

Choose another substrate -even synthetic if possible. More people could help you if you were to give the name of enzyme and substrate you are using now

Usually, in these cases, you should measure the initial reaction rate at different initial concentrations of product and substrates by separate, as it would for an enzyme which catalyzes an irreversible reaction. Then you can use a linear or nonlinear regression to estimate the kinetic parameters. On the Other hand, due to the spectrophometry does not work for your case, you could try with chromatography or others techniques. Due to, I don´t know which are substrates and product, I can not recomend you a technique for its quantification.

The research is difficult. Character of product and substrate must be compared carefully, other ways different from absorption value may be choosed.

The enzyme I am using is Cytosine deaminase; the substrate and products are either cytosine/ 5-fluorocytosine and uracil/ 5-fluorouracil respectively.

If your enzyme is not specific towards one substrate, then try some other substrates and see whether there is any reversible reaction occurs.

If this assay is the only assay works for you, you have no choice. Maybe you can try to find a formula with production inhibition to extract kcat and Km from the non-linear traces.

I insist, In first stage, you should try to measure the initial reaction rate at different initial concentrations of products and substrates by separate. I mean only S (Po=0) and then, only P ( So=0). Commonly, use another enzyme to remove the product is unnecesary . Since, during the assay to determine initial reaction rate (short time

1. MOst of the enzyme assays have been standerdized and are available in literature, If no standard assay method is available, anzyme assays are developed by determining first the optimum pH and temperature. Then the assays are run at different substrate concentrations for different time periods to determine the substrate concentration that yields maximum product in shortest possible time. It is usuualy the stage when system is fully saturated with substrate below the limit of substrate inhibition and initially there is virtually no reverse reaction. The reactants start reappearing only when substrate is consumed and its concentration becomes low. At equilibrium the concentration of rreactant is high. So u have to stop the reaction before that time just by determining the product formation.

Then run the assay by using the saturation level concentration of the substrate.

To determine Vmax and Km the standard assay is run for a fixed time period at optimumpH and temperature by using varying concentrations of the substrate usully in low substrate concentration range. You can always counter determine the maxiumum concentration of substrate. Ur last (highest) substrate concentration will be from 5-10 times higher than the KM determined from reciprocal plot.

So if standard assay for the enzyme is not reorted in literature, then you have to do all that analytical work.

2. In some cases where product concentration cannot be determined, we can determine enzyme activity by the rate of disappearance of subsrate

3. If both substrate and product cannot be determined, the rate of oxidation/reduction of a co.enzyme like NAD+ if involved can be determined for study of rate of reaction e.g. in case of lactate dehydrogenase reaction.

deaminase? very good, try IR (somewhere around 2000cm-1 if I remember correctly, should be easy to follow). Or try using something mild that would bind the amino base without denaturing the protein (has to be tested and screened, i guess...).

I have been using CALORIMETRIC methods for determining the kinetics of the reaction. I used Microcal iTC200 which are now commonly available. It works very well and is very easy simple and quick. Instead of absorbance you can use absorbance or generation of heat to follow the reaction. I am attaching our latest paper which will be very useful for you. Ertan H, Siddiqui KS et al. 2012 Biochimie 94:1221-1231. If you send me your email, I can send you the pdf of paper.

The Kintek Explorer software will allow you to determine kinetic constants by numerical integration from continuous reaction progress curves. I believe a student version can be downloaded at no cost. With this method, it is not necessary to use only initial rates or employ a second enzyme. The entire time course can be used, even until it reaches the final equilibrium. Multiple time courses collected at various substrate and product concentrations can be fit simultaneously.

Dear Amit ,

You have to use inhibition technique with standardization and validation it is little bit lengthy but approved method