I have a question in regard of the mixture of the apo- and holo-forms of an enzyme.
I know that the apoenzyme should be inactive while the activity of the holoenzyme is in correlation with the heme content. The activity increases as a result of the reconstitution of the enzyme with the heme.
However, is there any information about the influence of an apoenzyme or any other protein on the turnover rates of holoenzymes?
Hi there,
If the active enzyme is monomeric, there shouldn't be any effect apart from apo form being inactive and holo being active. If the enzyme is allosteric (ie. oligomeric) then the apo form should affect the activity depending on the occupancy level of the heme binding site (apo form being reluctant to allosteric transition).
I agree with what Dominique said, but would like to add a slight clarification to avoid confusion:
'Allosteric' describes a mechanism of action, whereas 'oligomeric' describes a structural form.
An oligomeric enzyme/protein may or may not be allosteric.
An enzyme with allosteric mechanism does not have to be oligomeric.
So activity of a holoenzyme may be affected either by an oligomeric partner, or by a structurally unrelated protein (effector).
besides allosterism, if you are trying to calculate specific activity and catalytic efficiency, then the apo form will affect these numbers.
Thank you for the answers!
I tested my samples which contained different ratios of apo- and holoenzymes. In my case, the apoenzyme did not influence the kinetic parameters of the holoenzyme. The holoenzymes from different samples had almost identical turnover rate.
Tarvi Teder for calculating turnover rate, what concentration of enzyme you are using? concentration of total enzyme or holoenzyme? if you are somehow calculating amount of holoenzyme in your sample and using it to calculate the turnover rate, turnover rate is not going to be different between different sample unless there is some form of allosterism. (provided your protein form oligomer)
Dhiraj Srivastava I have the apoenzyme without the heme and the holoenzyme with the heme. I determine the total concentration based on the absorbance at 280 nm and also the concentration of the holoenzyme based on the heme absorbance at 406 nm. As only the holoenzyme contributes to the activity and this activity is in correlation with the heme concentration, I use only the concentration of the holoenzyme in the kinetic measurements.
The concentration of the holoenzyme in a reaction is 10 nM.
Dhiraj, indeed. It was my initial idea to test whether the different amount of apoenzyme influence the turnover rate of the holoenzyme. In my case, the apoenzyme do not influence the turnover rate. However, I'll try to get rid of the apoenzyme or reconstitute the apoenzyme with heme (it is not worked out yet) in future to avoid incorrect results and the misinterpretation.
its my thought but I may be wrong. since you are using holoenzyme concentration in calculation, even your Kcat or specific activity should not be affected by excess apo (as long as they are not interacting). Km will not be affected even if you are using total enzyme concentration in calculation.
its good idea to work with as homogeneous sample as possible though.
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