They are as reliable, as careful your work is...

Steady state kinetics requires you to follow initial velocities (for most purposes). That implies taking your measurement under conditions where the starting conditions (the ones you set) are still prevailing. That period is characterized by a linear product formation rate (and naturally, a linear substrate consumption rate too).

To make sure this linear relationship is maintained, and hence the initial velocity period is not surpassed, one has to look at the progress curve ([P] or [S] vs. time) and take only the linear part.

This is easy to do in an assay with continuous monitoring of product consumption/substrate formation. With a discontinuous assay, you should take samples a several time intervals, follow the protocol to quantify the most convenient substrate/product, and plot the data. If linearity is not observed for at least the 4 initial samples. You should repeat your experiment reducing the time period or reducing the enzyme concentration.

You may save some work if you previously study under what period of time the rates are linear using the extreme values of variables to explore (i.e. is substrate and enzyme concentrations are to be varied, analyze the highest and lowest concentration of each variable, i.e. four conditions) and find the smallest time interval where the rate is linear for all four cases. Then you can keep all measurements within this time interval to make sure your rates are linear. This should be repeated for other variables you may wish to explore.

Now in pre-steady state kinetics, the experiment to do is called quench flow, and you must collect enough samples to dissect in detail the time period under analysis. There is no workaround in this case.

You may real more on this problems in the first two chapters en the classic book Enzyme Kinetics, by I. H.Segel.

Best wishes,

Rogelio

They are as reliable, as careful your work is...

Steady state kinetics requires you to follow initial velocities (for most purposes). That implies taking your measurement under conditions where the starting conditions (the ones you set) are still prevailing. That period is characterized by a linear product formation rate (and naturally, a linear substrate consumption rate too).

To make sure this linear relationship is maintained, and hence the initial velocity period is not surpassed, one has to look at the progress curve ([P] or [S] vs. time) and take only the linear part.

This is easy to do in an assay with continuous monitoring of product consumption/substrate formation. With a discontinuous assay, you should take samples a several time intervals, follow the protocol to quantify the most convenient substrate/product, and plot the data. If linearity is not observed for at least the 4 initial samples. You should repeat your experiment reducing the time period or reducing the enzyme concentration.

You may save some work if you previously study under what period of time the rates are linear using the extreme values of variables to explore (i.e. is substrate and enzyme concentrations are to be varied, analyze the highest and lowest concentration of each variable, i.e. four conditions) and find the smallest time interval where the rate is linear for all four cases. Then you can keep all measurements within this time interval to make sure your rates are linear. This should be repeated for other variables you may wish to explore.

Now in pre-steady state kinetics, the experiment to do is called quench flow, and you must collect enough samples to dissect in detail the time period under analysis. There is no workaround in this case.

You may real more on this problems in the first two chapters en the classic book Enzyme Kinetics, by I. H.Segel.

Best wishes,

Rogelio

Yes, it is reliable. Since:

1) Enzyme, substrate, and ES complex are at equilibrium.

2) Substrate concentration is very much larger than enzyme concentration

3) The overall rate of the reaction is limited by the breakdown of the ES complex to form free enzyme and product.

4) The velocity is measured during the very early stages of the reaction so that reverse reaction is insignificant.

See Enzyme Kinetics, I H Segel.

I like Rogelio answer, but I would pay more attention to the concentration changes rather than the elapsed time.

I do agree with the statement of Rogeilo. I want to add that consideration of enzyme concentration as well as purification state is very important while working on the kinetics of enzyme. Also, nature of assay depends on whether the enzyme of interest is membrane bound or soluble one.

Researchers interested in working with 'Enzyme purification & Characterization' may survey the following research articles thoroughly:

(1) Mishra, S., Sapola, B.M., Clark, M.A. and Vijayaraghavan, S. (2011). Evolutionarily conserved glycogen synthase kinase-3 in sea urchin spermatozoa is regulated by egg-derived factors. Biotechnology, Bioinformatics and Bioengineering 1: 99-108

(2) Mishra, S., Shenoy, S P.R., Huang, Z., and Vijayaraghavan, S. (2003). Binding and inactivation of the germ cell specific protein phosphatase PP1g2 by sds22 during epididymal sperm maturation. Biology of Reproduction 69: 1572-1579.

(3) Mishra, S. and Kamisaka, Y. (2001). Purification and characterization of thiol reagent- sensitive glycerol-3-phosphate acyltransferase from the membrane fraction of an oleaginous fungus. Biochemical Journal 355: 315-322

(4) Pathak, N., Mishra, S. and Sanwal, G.G. (2000). Purification and characterization of polygalacturonase from ripening banana fruit. Phyotochemistry 54: 147-152.

(5) Kamisaka, Y., Mishra, S. and Nakahara, T. (1997). Purification and characterization of diacylglycerol acyltransferase from the lipid body fraction of an oleaginous fungus. Journal of Biochemistry 121: 1107-1114

In my opinion and belief, these papers would certainly be helpful to researchers working in the relevant area to justify the protocols in accordance to the cellular status of the enzyme. further queries in this relevance are welcome.