Although it is a lot of work, there really is no substitute for a thorough examination of the concentration-rate relationship for all 3 of the substrates, preferably as a three-dimensional matrix of concentrations (not so hard to do if you work with microplates instead of with cuvettes). You may be working in a region of substrate concentration at which the apparent Km for the one varied substrate is very high.

Watch out for the possibility that you are seeing a background rate of spontaneous reaction, which can be tested by (1) leaving out the enzyme and (2) leaving out each of the other substrates one at a time.

Hi there,

It's difficult to figure what is going wrong in your experiments as you don't give any details about the range of concentrations you used (forthe substrates and the enzyme) and the relationship between initial velocity and the varying concentration.

Although it is a lot of work, there really is no substitute for a thorough examination of the concentration-rate relationship for all 3 of the substrates, preferably as a three-dimensional matrix of concentrations (not so hard to do if you work with microplates instead of with cuvettes). You may be working in a region of substrate concentration at which the apparent Km for the one varied substrate is very high.

Watch out for the possibility that you are seeing a background rate of spontaneous reaction, which can be tested by (1) leaving out the enzyme and (2) leaving out each of the other substrates one at a time.

Hi Adam and Dominique. Thanks for your replies. At this stage I'm going to have to acknowledge you in my thesis! 

I''ll edit my question to include details but for the moment here they are: I use 1mM NADPH, 1mM ATP and a series of 5 concentration of the acid to be reduced. The rates I see are strange. It looks like there is an initial rate followed by a much faster rate and then a drop. I cannot seem to get a straight line when I plot the reciprocal.  

Hi again,

1mM NADPH is giving a huge signal at 340nm (OD=6.22). Are you sure to have the suitable spectrophotometer to run quantitative analyses starting from such high OD?

Hi Dominique unfortunately the enzyme is extremely inefficient (at the moment) and requires high levels of NAPDH. Our machine seems fine with this concentration.  

Your description of the kinetics is as if there is a pre steady state phase (what you call initial rate) followed by steady state (much faster rate). Have you tried to use the velocity data from this latter phase for plotting versus substrate concentration? As it is abouta three substrate kinetics, it might be that the steady state takes some time to be reached.

For my personal knowledge, what spec are you using? I've never used a spec giving a stable and reliable reading when OD is above 3...

I agree with Dominique Ligier. You must check wether the absorbance is linear with NADPH concentration at 1 mM NADPH. When the log converter is saturated you will get, even for a linear reaction on time, an initial slow rate rate and an acceleration as long run into the correct absorbance range (generally lower than 3.0). If you need to use such high NADPH concentration you must used cuvettes with path lenghts lower than 1 cm.  

Hi Harry,

   Very constructive discussion up there. Let me add my two cents to this fantastic chain of responses.

1) The first thing you would have to make sure is that enzyme should be present in catalytic amounts. I noticed your point that the enzyme is lousy and requires high substrate/cofactor to show activity. Under such circumstances people often make the mistake of using non-catalytic amounts of the enzyme to get activity. That would make the kinetics non-MM, making it difficult to interpret the results. Second, make sure you are working in a range of enzyme concentration where the activity is linear as enzyme conc. is increased. Third, make sure that you are assessing activity when less than 5% of the substrate is converted to product.

2) I notice that you talk about a lag before achieving steady state. There could be several reasons for that one among which is the existence of the an inactive enzyme form. Try initiating the reaction with enzyme preincubated with NADPH and ATP, respectively to see whether the lag vanishes. An alternative approach would be to have the enzyme in the common mix and try initiating the reaction with either NADPH or ATP addition. If you notice the lag vanishing in the reaction with ATP, you can be sure that NADPH is responsible for flipping the enzyme from its non-active form to its active form.

3) I assume that the third substrate is an organic acid. Did you ensure that the pH of the solution is not disrupted when you titrate the substrate.

4) metabolites like ATP usually bind to their enzymes as Mg-ATP. In case this is the case with your enzyme, you would have to incorporate enough magnesium to make sure all the ATP is in its magnesium form. 

I hope these suggestions, along with others above, help you get over the bottleneck.

Best Wishes,

Bharath

I agree 100% with Bharath about ATP and magnesium ion. If you simply vary ATP concentration without taking care about what is the true substrate and thinking about stability constants for Mg++-ATP complexes you are bound to run into trouble (see e.g. papers from Stanley Ainsworth on Yeast pyruvate kinase - Biochem J 1970s).

However, I don't think you have reached that point yet. As the others have pointed out, nobody would think of measuring even at OD 3. At OD 3 only 0.1% of the light is getting through! At OD 6 it will be 0.0001% !!!   Your PM tube or photodiode will be staring out there, waiting for the next photon to come by!  Your strange progress curves are almost certainly a photometric artifact. You MUST find conditions where you get a linear initial rate and where that rate is clearly and accurately proportional to the amount of enzyme you add. It seems unlikely that an NADPH enzyme needs 1mM and that impression could be down to the fact that you are not really seeing the rates at all.  (What sort of trace to you get with 0.1mM, which corresponds to a perfectly manageable OD of 0.62?) However, if we assume it really does need 1mM, then you could decrease your massive OD by going out to a longer wavelength instead of the peak at 340nm. You could also use cuvettes with a 1 or 2 mm light path instead of 1 cm.     By the way, is it NADH or NADPH?  In your initial question you said NADH and then later NADPH.

Eventually, if you want complete mechanistic information, you do need, as Adam said, to do a systematic 3D matrix study with variation of all three substrates (see Biochem J 108, 409-419 1970). However, if you just want 'apparent Km values' you can fix two substrate concentrations at a time nad vary the third. Depends on what you need.