I have obtained a graph which was plotted by using the equation v= kcat[S]/(Km + [S] + [S2]/Kis) .
Substrate concentration varied from 0.1-5mM. But when iam going for data points lower than 0.1mM (10-100uM :not shown in graph), initial velocity is very high at 10um and it keep on decreasing as substrate concentration reaches 100um.
After 100uM, initial velocity is increasing with increased substrate concentration(0.2-5mM).
Even though data was fitted properly with above equation what might be the possible reason for this anomalous behavior before 100uM and after 0.2mM substrate concentration.
Please give your suggestion..
Considering most points are along the curve, it is likely that the value at 10 uM is erroneous. I would repeat that data point or do a q-test to see if you can eliminate it as an outlier. In addition, it would be useful to know the enzyme concentration in the experiment and to know if co-factors are necessary. For example, in DNA polymerses when we use high nucleotide concentrations we also need to add additional catalytic metal ion co-factors as many of the ions get sequestered by the excess substrate, which in turn looks like substrate inhibition.
Hi there,
I agree with Andrew. Repeat the experiment to check the validity/repeatability of your data (especially as the two results you mentioned look very much alike the 2 following in the serie...).
If it turns out that your strange result is reproducible, then consider whether your substrate is pure. I'm thinking that perhaps it consists of at least two different compounds, both of which are substrates. There is a low-Km substrate and a high-Km alternate substrate that acts as a competitive inhibitor with respect to the low-Km substrate. This situation might lead to the complex Michaelis plot.
Thank you all...
Iam doing indirect assay coupled to pyruvate kinase/lactate dehydrogenase where oxidation of NADH was measured at 340nm.
I have repeated experiment more than 5 times still I used to get the same result.
Reaction mixture contains....
Enzyme (kinase ) : 0.65uM
Substrate : 0.1-5mM
Mgcl2 :2mM
KCl : 100mM
ATP : 0.75mM
Phospho enol pyruvate (PEP) : 0.3mM
NADH : 0.2mM
Pyruvate kinase : 0.4U/ul
Lactate dehydrogenase : 1.2U/ul
@Andrew
I have added all components according to a reference paper...
In addition to Pyruvate kinase my enzyme(kinase) also requires both Mg2+ and K+ for the activity.
I will also try Q-test.
@Amit
My reaction is reversible and ratio of the enzyme (0.65uM) to substrate(0.1-5mM) is also high.I will try to fit my data Haldane equation.
@Adam
My protein transfers phosphate group from ATP to substrate and based on the preliminary results both substrates have slightly different Km values.
Km for S1 : 0.32mM
Km for S2 : 0.2mM (ATP)
When I have varied S2 and kept constant S1 and MM graph is perfectly fitted but it is not possible in vice versa.
I was not referring to the two nominal substrates, ATP (S2) and whatever is being phosphorylated (S1). I was referring to the possibility that S1 might be impure, containing at least 2 substances that can act as substrates but that have very different Kms.
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