Dear Micheal and Matthew: Thank you for your inputs on the structure-function analysis data. As pointed out by Micheal - the change in Km (For Met to Ala mutation) is not much. Could this be intepreted simply that the mutation does not change the affinity of the enzyme for the substrate because a smaller side chain (ala) would not obstruct, if not facilitate, the placement of the substrate. However, a slight increase in Km would suggest a larger side chain (met) is needed to guide and position the substrate optimally? This is further supported by a decrease in Vmax. I would assume, the Vmax values calculated at lower concentrations of substrate, as used in kinetic analysis, are generally not accurate (as opposed to that Km values at lower concentrations of substrate are considered fairly accurate). However, the general trend of the kinetic analysis showed that the Vmax for the Met to Ala mutation decreased significantly. To verify this, independant assays ( as a separate experiment) were performed for the wild type and mutant under identical conditions with saturation concentration of the substrate. Here, the differences in the Vmax for the mutant and wild type were magnified as indicated by a 15 % residual activity for the Met to Ala mutation. On the other hand, Matthews seems to suggest that the differences are not significant. Would a 85 % loss in activity at saturating concentration (6.7 fold decrease) considered insignificant? This could be more to do with the sensitivity of the assay. However, since the experiments were conducted under identical conditions, wouldn't these differences be real and significant? I truly appreciate your time and valuable suggestions :> Sincerely, Deepak .
On Thu, Feb 18, 2010 at 12:34 AM, R. M. Garavito <rmgarav...@gmail.com>wrote: > Deepak, > > The changes you see are not huge and could be in the noise. > > > 1. Km increased by 0.5 fold -- not much of a change > > These seem to be a little inconsistent as Vmax = kcat*[E]: > > > 2. Vmax decreased by 3.5 fold > > 3. Kcat decreased by 4 fold > > These are not too different. > > > 5. Activity at saturating concentration of substrate - only 15 % of the > wild type. (a 6.7 fold decrease) > As activity at saturating concentration of substrate is Vmax, why is it so > much lower? > > Did you use the same enzyme concentration for the experiments (most protein > assays are not particular accurate and are variable)? Did you benchmark [E] > with A280 measurements? > > Did you repeat the kinetic analysis of the WT enzyme along with the mutant > (using the same buffers and reagents)? > Even slight differences can arise between two kinetic trials a month or two > apart just due to differences in buffers and reagents (age, slightly > different pH, etc). > > Were the temperatures of the assays the same (did you use a thermostated > cell)? > > Moreover, in choosing the amount of enzyme to use, it must be in the "valid > range" to get good data. This means that Vmax = kcat*[E] must hold. The > valid ranges for the WT and mutant enzymes may not be the same. > > At this point the mutant seems not to have made much of a change. Repeat > the analyses back to back to get more data. > > Good luck. > > Michael > > ****************************************************************** > *R. Michael Garavito, Ph.D.* > *Professor of Biochemistry & Molecular Biology* > *513 Biochemistry Bldg. * > *Michigan State University * > *East Lansing, MI 48824-1319* > *Office:** **(517) 355-9724 Lab: (517) 353-9125*** > *FAX: (517) 353-9334 Email: garav...@msu.edu* > ****************************************************************** > Matthew Merski to me show details 5:18 AM (3 hours ago) No, these data show only small changes in the activity of the enzyme (less than an order). Typically you want to see something with at least 10^4 fold effects to be sure it matters and really you should see something like loss of 99% of the activity to start arguing for an effect. The Met to Ala mutation is probably perturbing the active site a little since the shapes are different, but not doing much else. The differences you see are not large enough that you can't honestly rule out experimental error as their cause, especially if you are using literature data for the WT values or the two measurements were performed by two different people. Methionine acting like a nucleophile would be unlikely enough that I would call it impossible. Matthew Merski UCSF Shoichet Lab - Show quoted text - > > On Feb 17, 2010, at 10:43 AM, Deepak Oswal wrote: > > Dear colleagues: > I am trying to interpret the results of the substitution of a Methionine > with Alanine. Following is the kinetic data on the mutation - > 1. Km increased by 0.5 fold > 2. Vmax decreased by 3.5 fold > 3. Kcat decreased by 4 fold > 4. Kcat/Km decreased by 10 fold. > 5. Activity at saturating concentration of substrate - only 15 % of the > wild type. > Is it possible to conclude from the data that the methionine is involved in > stabilization of the transition state (the methionine is located inside the > putative active site; we do not have a structure of the enzyme-substrate > complex)? Is this even possible atall? > Although impossible, on second thought, given the ability of the > micro-environment to alter/bring down pKa s, is there any instance or > possibility that a methionine could or has acted as a nucleophile? > In addition to all other routine precautions to avoid experimental errors, > a CD analysis of the mutant and wild type has been performed and there are > no obvious differences in the spectrum. The wild type and mutant enzyme show > almost identical size exclusion profiles. > I would appreciate any suggestions or comments. > Sincerely, > Deepak > > >
