For sure, you will need to collect data at lower substrate concentration to determine what is going on for processed substrate. One should also consider how the "initial rates" are measured. Ideally, the amount of substrate depleted during the initial rate measurement should be less that 5%. When it is larger than this, or when the product is a strong inhibitor of the enzyme, complicated or even artifactual results can be obtained. We have seen similar, time-dependent kinetic behavior for an enzyme that is strongly allosterically inhibited by its product. For complex kinetic behavior, it may be necessary to have a finer grid of measurements to allow for accurate data fitting.
As others have pointed out, linear transforms of the Michaelis-Menten equation are statistically flawed without applying proper y-weighting. It is much better to do nonlinear least squares fiting of rate vs. substrate data. For true product inhibition, there are several possible models that could be considered, based on the behavior of the data. Consultation with an enzyme kineticist might be warranted for for complex behavior. Roger Rowlett On Fri, Jun 18, 2021, 3:53 AM Harmer, Nicholas <[email protected]> wrote: > Dear Prem, > > > > I agree entirely with Tristan’s conclusion that the processed substrate is > also acting as an inhibitor at higher concentrations. You would need to run > an experiment with a wider range of concentrations used (especially lower > concentrations) to get a better feel for the range of the reaction. There > is a well described substrate inhibition equation (see e.g. > https://www.graphpad.com/guides/prism/latest/curve-fitting/reg_substrate_inhibition.htm) > that you can try. I have a recent chapter on experiment design covering > such cases ( > https://www.researchgate.net/publication/331806855_Reaction_Chemical_Kinetics_in_Biology) > that I can share with you if you need. > > > > I would strongly recommend to avoid the Lineweaver-Burk plot to calculate > your Km and kcat. There are known issues with this (especially, as in your > image, overweighting the lowest rate and usually least accurate point). > Better is to fit directly to the equation with non-linear fitting, for > example in *R*. This will also give you a better estimate of the error > and confidence intervals. > > > > Hope this helps, > > > > Nic > > > > *From:* CCP4 bulletin board <[email protected]> *On Behalf Of *Tristan > Croll > *Sent:* 18 June 2021 08:19 > *To:* [email protected] > *Subject:* Re: [ccp4bb] Enzyme Vmax and Km > > > > Hi Prem, > > > > The immediate problem here is that the curve for the processed substrate > simply cannot be described by simple Michaelis-Menten kinetics. Assuming > the assay has worked as expected, the declining rate with increasing > substrate concentration suggests to me that this substrate also acts as an > allosteric inhibitor, so assays at high [substrate] will make it *look* > like the unprocessed substrate is preferred even though the processed one > is cleaved faster by the uninhibited enzyme. > > > > Hope this helps, > > Tristan > > > On 18 Jun 2021, at 05:05, Prem Prakash <[email protected]> wrote: > > Dear all, > > Sorry for this off topic. I am working on an enzyme that has an > exonuclease activity. The enzyme preferentially cleaves an unprocessed > substrate at a faster rate than the processed one (known by qualitative > analysis). Recently, I calculated the Vmax, Km and kcat of the enzyme for > unprocessed substrate which are 18.2 pmol/min, 182 nM and 7.1 sec-1 > respectively. However, the Processed substrate has apparently a lower range > of Km (not calculated) as reflected from the curve (because the same > increasing concentration range which is used for unprocessed, shows a steep > decline in the initial velocity of the enzyme with processed substrate. > > The latter suggests that Km is way lower than expected. In this case, the > question is, if the Km of processed substrate is way lower than the > Unprocessed, how can we see a faster rate with the former substrate than > later. i.e lower Km and slower rate of cleavage. If it's possible please > give some insights. I have attached the plot comparison between two kinetic > assays. > > > > With kind regards, > > > > Prem > > > > > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > <https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2FWA-JISC.exe%3FSUBED1%3DCCP4BB%26A%3D1&data=04%7C01%7CN.J.Harmer%40exeter.ac.uk%7Cc87605fa4f1643fcd1ab08d932296088%7C912a5d77fb984eeeaf321334d8f04a53%7C0%7C1%7C637595975551764397%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=xlQ3OZDw16skYx2%2B0VjJCfH1%2BiyfjG7kYwiuv6dZ5aw%3D&reserved=0> > > <Picture1.png> > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > <https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2FWA-JISC.exe%3FSUBED1%3DCCP4BB%26A%3D1&data=04%7C01%7CN.J.Harmer%40exeter.ac.uk%7Cc87605fa4f1643fcd1ab08d932296088%7C912a5d77fb984eeeaf321334d8f04a53%7C0%7C1%7C637595975551774392%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=wI7QEgT3dWPiIR7GmEPH1y5Hk6TBjkhEX6reRwinYEI%3D&reserved=0> > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
