Dear Tristan and all my ccp4bb members, Thank you so much for your valuable suggestions into the enzyme kinetics issue I have been facing. It gave me new insights to design my experiment and I started working on those inputs. Thank you all once again. It's very helpful.
With kind regards Prem On Fri, Jun 18, 2021 at 2:19 AM Tristan Croll <[email protected]> wrote: > 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 > > <Picture1.png> > > -- With kind regards, Prem Prakash Ph.D. Research Scholar Protein Crystallography Lab. Dept. of Biosciences and Bioengineering Indian Institute of Technology Bombay ######################################################################## 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/
