Re: [ccp4bb] Substrate density visible in relatively active mutant

[Sudipta Bhattacharyya]

Hi Seema,

I guess the turnover number of the mutant enzyme is considerably low
compared to the wild type one, as well as in the crystallization condition.
It might be because the energy barrier of the transition state could be
much higher for the mutant enzyme in the given crystallization condition
compared to the wild type version.

Best,
Sudipta.

Sudipta Bhattacharyya
Postdoctoral fellow,
Department of Molecular Biosciences,
University of Texas at Austin,
Austin, Texas, USA.

On Fri, May 26, 2017 at 3:48 PM, Seema H Irani 
wrote:

> I crystallized a PLP dependent enzyme and wanted to capture the enzyme
> substrate complex, however due high activity of the enzyme I could see no
> density at the active site upon soaking with the substrate. I hence made a
> bunch of mutants of the active site residues and one of them showed density
> consistent with the substrate in some of the molecules in the asymmetric
> unit. Surprisingly however when I measured the activity of this mutant it
> had 30% of the wildtype activity I am wondering why is it still possible
> for me to see substrate density for this mutant?
>
>
>

Re: [ccp4bb] Substrate density visible in relatively active mutant

[Parthasarathy Sampathkumar]

Hi Seema,

Couple scenarios plausible (i) mutant is "super slow" compared to WT that
crystallization was setup prior to any significant reaction and / or (ii)
components, as well the pH, of crystallization condition made the reaction
even more slower. Of course, if the reaction has partially occurred, you
are likely to observe occupancy less than 1. These plausible only
scenarios!!

Hope this helps,
Partha
On Fri, May 26, 2017 at 4:59 PM Seema H Irani 
wrote:

> I crystallized a PLP dependent enzyme and wanted to capture the enzyme
> substrate complex, however due high activity of the enzyme I could see no
> density at the active site upon soaking with the substrate. I hence made a
> bunch of mutants of the active site residues and one of them showed density
> consistent with the substrate in some of the molecules in the asymmetric
> unit. Surprisingly however when I measured the activity of this mutant it
> had 30% of the wildtype activity I am wondering why is it still possible
> for me to see substrate density for this mutant?
>
>
>

[ccp4bb] Substrate density visible in relatively active mutant

[Seema H Irani]

I crystallized a PLP dependent enzyme and wanted to capture the enzyme
substrate complex, however due high activity of the enzyme I could see no
density at the active site upon soaking with the substrate. I hence made a
bunch of mutants of the active site residues and one of them showed density
consistent with the substrate in some of the molecules in the asymmetric
unit. Surprisingly however when I measured the activity of this mutant it
had 30% of the wildtype activity I am wondering why is it still possible
for me to see substrate density for this mutant?