Is your enzyme monomeric or multimeric? What time exactly does it mean when you say unprocessed substrate versus processed?
What are your error margins (no error bars evident on your plots)? Depending on your errors, your second plot is not a 'steep decline' but instead could be a constant line. So, if your affinity for processed substrate is higher, it does not have to mean that catalytic efficiency will also be higher For example RNA polymerase have to eventually let go of the promoter region in order to elongate, but if the promoter binds too tightly they will instead abort translation and fall off. Nature is complicated :) In general it would help us a lot to help you if we understand the details of your issue. There is also a common issue that can be encountered with weakly associated multimeric enzymes that act on polymeric substrate (in your case, DNA) - namely when you add more substrate than enzyme the monomers of enzyme can become 'smeared' along the DNA and this results in very odd kinetic behavior. Artem On Fri, Jun 18, 2021, 12:05 AM Prem Prakash <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 > ######################################################################## 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/
