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 <CCP4BB@JISCMAIL.AC.UK> On Behalf Of Tristan Croll Sent: 18 June 2021 08:19 To: CCP4BB@JISCMAIL.AC.UK 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 <prem...@gmail.com<mailto:prem...@gmail.com>> 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 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/
