Hi everyone,

I'm a chem PhD student desperately trying to finish! I'm analysing and writing up my final experiments but have been having considerable problems trying to settle on a correct method of analysis.

Basically I've prepared several metal complexes that catalyse the hydrolysis of an ester. I want to determine the catalytic rate constant (kcat, Vmax) for each complex along with the Michaelis constant (Km) for the substrate-catalyst (ie ester-complex) interaction. Following conventional Michaelis-Menten kinetics you'd measure the rate of experiments with the [catalyst] constant and small and with increasing [substrate]. You can then determine kcat and Km by plotting the rate against the [substrate] using the classic Michaelis-Menten equation:

Y = Vmax*[substrate]/(Km + [substrate])

However, I've had to do my experiments by keeping the [ester] constant and small and increasing the [complex]. You can't simply substitute [substrate] for [catalyst] in the MM equation because at [substrate]=[catalyst]=0, Y will = 0. When there's zero ester to hydrolyse this is correct (because there won't be any hydrolysis at all), but when there is no catalyst present the ester will still slowly hydrolyse in solution (ie the uncatalysed rate, kuncat, Vuncat) and therefore Y does not equal 0. This uncatalysed rate is a constant - this reaction goes on in solution even when the catalyst complex is added.

The data I've collected fits with the MM method, but this is clearly not correct because the graph shouldn't pass through 0,0. (Basically my data is exactly the right curved shape but the graph starts above 0 at the Y-intercept.) I've found some linearised versions but my data does not fit with these.

Is there something I can do to the traditional MM equation above to account for this uncatalysed rate for [complex] = zero? Something like:

Y = (Vmax*[complex]/(Km + [complex])) + Vuncat

I don't think this is quite right but I'm sure there's a simple way of doing this.

Thanks so much for any advice anyone can give!

*Bump*

Can anyone help Ceywood? This post is making my head hurt!

Hi Reenie,
thanks for taking the time to have a think about this - it's making my head hurt too! It's the derivation that's the killer but I think I may have come up with a solution during that 2am peak PhD brain function time last night actually! Got to show it to a few people but if it's confirmed as a plausible analysis method then I'll post it back here :)

Thanks again!

Hi Ceywood - I didn't even try to understand it (as I know it is way beyond me), I just tried to get the gist of your question - and failed! Good luck :)