I’m working with a rather complicated ping pong, bi-bi double displacement reductase enzyme that oxidizes NADH and then reduces a substrate. Interestingly, the enzyme has intrinsic NADH oxidase activity in the absence of the substrate. Of course, upon addition of substrate, the velocity of the reaction increases and NADH is consumed more rapidly. The intrinsic activity introduces a baseline velocity and is complicating our kinetic analyses a bit. Complicating it even further is a noted double substrate inhibition pattern (apparently not uncommon with ping-pong mechanism enzymes). We’ve run a full course of analyses, varying the NADH as well as the substrate and we're now trying to fit the data to an appropriate equation. My question is: How do I take the ‘intrinsic activity’ of the enzyme into account (if I need to at all)? Can anyone recommend an equation and appropriate software that can do this? We currently have SigmaPlot, GraphPad, and Enzfitter (for the double substrate inhibition model). Thanks in advance for any guidance, ideas, and/or discussion.
Hi there,
The issue is to strictly identify the 2 activities contributing to NADH oxydization. NADH oxydase activity results in hydrogen peroxyde formation. Then from a single reaction mixture you should measure the decrease of absorbance at 340nm which gives the total activity and in parallel measure H2O2 production which is specific of NADH oxydase activity. The difference between the two gives you the reductase activity.
An other possibility would be to work in anaerobic condition to prevent the oxydase activity which uses O2 as a substrate.
Make sure the NADH oxidase activity is being catalyzed by your enzyme, not by contaminating enzyme, and also that it is not just the spontaneous (no enzyme) rate of NADH oxidation.
If it really is due to your enzyme, decide whether it is large enough to affect the results when both substrates are present. If not, ignore it. If it is uncatalyzed, then just subtract it from all the catalyzed rates.
I don't know the kinetic rate equation for this system with the extra NADH oxidase activity. Maybe Segel has it in his big book. If you have to derive it yourself, it will be a complicated business.
Without the NADH oxidase activity, the rate equation is:
v = Vmax[A][B]/{KmA[B](1+[B]/Kib) +KmB[A](1+[A]/Kia)+[A][B]}
You can also get the kinetic constants by numerical integration of the progress curves, such as by using Kintek Global Kinetic Explorer. The model would look something like this (A is NADH and B is the other substrate):
E + A = EA = E + P (NADH oxidase activity)
EA + B = [EAB = EPQ] = E + P + Q (the part in brackets should probably be omitted)
EA + A = EAA (substrate inhibition by NADH)
E + B =EB (substrate inhibition by second substrate)
The program can be used to get a global fit to a set of progress curves (not just initial rates) collected at different A or B concentrations at a fixed E concentration and a fixed concentration of the other substrate. It takes a lot of persistence to get solid values for Kms and kcats this way, but the results usually match pretty closely to the initial rate method. You can also take product inhibition into account with this method.
I'd strongly agree with the answers above. Between them they cover the possible scenarios very well.
Kintek Explorer is a great tool, although it is as mentioned above hard work to get right.
Good reference for the equation is "Fundamentals of enzyme kinetics" (Cornish-Bowden, 2012, 4th ed.).
Hi Jesse,
I once had an issue very similar to what you describe. I measured "Background" NADH consumption in the absence of Substrate, but when substracting this activity from activities in the presence of substrate it yielded odd curves. My solution was to measure the NADH consumption at 340 nm and taking samples from the reaction and analysing substrate and product concentration using a gc. This showed that the electron-coupling efficiency was 100% with good Substrates (each NADH used resulted in one Produkt molekule). Thus the Background NADH consumption dissappeared in the presence of a substrate. You can see it as water and the other substrate being competing substrates, when they are both present the real substrate is highly preffered, (kcat/KM)Substrate/(kcat/KM)water>>1, so the activity with water ("background" activity) can be neglected, but when there is no other substrate present water will be oxidised to H2O2 as mentioned above. Thus try to correlate the NADH consumption with the substrate conversion and/or product formation determined with an alternative method!
Good luck!
You need to separate both activities. 1. Make sure the NADH oxidase activity is bona fide. NADH can be unstable at low pH, so make sure the NADH loos is catalysed by the protein. 2. Make sure your protein is pure. If you have contaminants, the NADH oxidase may be a contaminant enzyme. 3. Once the above is answered, if this activity is indeed catalysed by your enzyme, analyse the kinetics of the NADH oxidase activity. Redox reactions require a pair, identify the electron acceptor, it could be oxygen, but it could be some other compound, like the buffer. 4. Once your oxidase activity has been characterized, you can start with the other activity. For each individual measurement, you need to make sure you are in the linear portion of the progress curve and then subtract the oxidase activity rate from the NADH consumption rate, which you may measure in a parallel control or calculate from the previous characterization. Best wishes Rogelio
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