Do you know the kinetic mechanism of your enzyme and the corresponding rate equation? If so, then you can attempt to use a nonlinear least-squares regression computer program to fit the initial rate data collected for a matrix of substrate concentrations to the equation in order to obtain values for the kinetic parameters. I have used Grafit (Erithacus Software) for this purpose.

If you are seeing substrate inhibition, then the rate equation can be suitably modified to incorporate this into the mechanism. An important point is that you collect initial rate data while varying the concentrations of all the substrates, not just one.

You need to deeply learn chemical kinetics to build the right kinetic model. This is a really hard task even for very experienced kineticists. Do not rely upon any software and use standard schemes. A software can't solve the problems, the humans solve the problems. I agree with Adam: "Do you know the kinetic mechanism of your enzyme and the corresponding rate equation?" You have to collect your data purposely, but not just to have "some data." These data might be useless

Thank you all for replying. I know the mechanism of my enzyme. it is a monooxygenase that is a flavoenzyme, got reduced by NADPH and then upon reacting with molecular oxygen, it oxidize L-lysine to hydroxylated Lysine. 

I am working with a mutant of my enzyme.

In the assay I am measuring the amount  of product (L-hydroxy Lysine) varying each substrate at saturation level of the other. 

I have two sets of data for each: "attached"

the wild type enzyme, normally shows substrate inhibition behavior and the data published has a good fit to simple substrate inhibition equation  : 

v =(kcat[S]) / (KM +[S]+[S2 ] / Kis)

the mutant shows the same behavior but at higher concentrations of each substrate, so I am using higher concentration of each for saturation.

the data could n't be fitted to the same equation!!

that's why I was trying to explain this or find a different way to understand the mechanism.

Dr. John H.T. Luong 

Thank you for your contribution. I am going through the link you kindly suggested.

 Dear Heba and John,

As far I understand this is typical two steps catalytic process: 1) Reduction of an enzyme by NADH;2) oxidation of lysine by O2. Depending on conditions/concentrations the rate limiting step might be either 1 or 2. This results in a very complex kinetic model. Dioxygen might be the third (in addition to NADH and lysine) substrate/regent in this system. My suggestion is to check the effect of dioxygen concentration  (partial pressure) on the reaction rate. The simplest would be to carry out the reaction under air and under pure (100%) O2. 

This enzyme has a ter-reactant mechanism, with the 3 substrates being NADPH, O2, and L-lysine. The kinetic rate equations for such mechanisms are pretty complex, and there are several possibilities. Since you have only varied NADPH concentration at "saturating lysine" and lysine concentration at "saturating NADPH)," in my opinion you have not really got enough data to analyze the substrate inhibition mechanism properly. Putting aside the lack of any data on the effect of O2 concentration (experimentally challenging), you should measure rates for a matrix of NADPH x lysine concentrations. This should always be done, but it is especially important when you have substrate inhibition because you can't really have a "saturating" concentration of the substrate that inhibits. You can never reach Vmax (the definition of saturation) because of the inhibition.

Dr. Adam B Shapiro, 

Yes, that what I was doubting considering reaching Vmax value with this sort of inhibition. I was trying to understand how this was done and published for the wild type.

and I can see that I have this inhibition only when I am monitoring the product formation, when I am using oxygen consumption as the assay. I don't see that inhibition. this should be related to the product or substrate binding still enhance the oxygen consumption unproductively forming other oxygen species. that's why I exclude having a limitation of oxygen during the reaction that may affect the maximum rate. 

So my concern is even if I can determine kinetic parameters using the data I presented, it won't be a real reflection of the real activity of the mutant! 

Is there other way I can refer to these data with?

and about the matrix, you mean varying NADPH (for example) at different suboptimal, optimal and higher L-lysine concentration and vice versa, right!!

So, in that case I am not sure how to combine these data that I will have! should I compare the parameters I have only to get what best represent my enzyme kinetically!

Thank you

appreciate the discussion

If I understand correctly, you see the substrate inhibition when you measure product formation (What is the product being monitored - NADP?) but not when you measure O2 consumption? Ideally you would get the same result in both cases, but since you don't there is the concern that some nonproductive O2 consumption is happening.

This could be a technical problem rather than a biological one. It is more difficult to accurately measure substrate (O2) consumption than product formation. The reason is that there is a small change from a high background when measuring substrate consumption, but a large change from a low background when measuring product formation. I would have more confidence in the product formation measurement. But keep in mind that there may really be some unproductive O2 consumption. This may be especially apparent when the lysine concentration is relatively low and the O2 concentration is relatively high.

Meanwhile, the fact that you are able to measure O2 consumption tells me that you have the capability to vary O2 concentration in the assay. Since you can vary the concentrations of all 3 substrates, you will have to do so in order to measure all of the kinetic constants in the rate equation, whatever it is, no shortcuts. This means performing a 3-dimensional matrix of initial rate measurements. For example, if you test 5 concentrations of each substrate, you will have 5 x 5 x 5 measurements to make.

To obtain the kinetic constants (KmA, KmB, KmC and whatever substrate Kis are in the rate equation, plus the substrate inhibition constant(s)) you will need to use the rate equation that is appropriate for the kinetic mechanism. Then you can compare the kinetic constants for the mutant enzyme with the kinetic constants for the wild-type enzyme.

If this sounds like a lot of work, it is.

Dr. John H.T. Luong 

thanks so much for the helpful suggestions. it keeps me learning. for that, May I ask How you were able to fir the data and why you used this particular equation, How to judge that when you have data like this!

Dr. Adam B Shapiro and Dr. John H.T. Luong 

I have re-done the experiments again at to monitor the oxygen depletion. it was very limited in short time, which was interfering with the results I have. I was having substrate inhibition as wells as more anaerobic system as the substrate concenteration and the time of reaction increase.

I repeated product formation assays, and I have a really good results this time, I have considered well shaking and ventilation of the reaction system within a descent reaction time.  

Thank you all for giving explanation for everything

Thank you Dr. John Luong. for this clarification. 

I appreciate your help.