JiscMail Logo
Email discussion lists for the UK Education and Research communities

Help for CCP4BB Archives


CCP4BB Archives

CCP4BB Archives


CCP4BB@JISCMAIL.AC.UK


View:

Message:

[

First

|

Previous

|

Next

|

Last

]

By Topic:

[

First

|

Previous

|

Next

|

Last

]

By Author:

[

First

|

Previous

|

Next

|

Last

]

Font:

Proportional Font

LISTSERV Archives

LISTSERV Archives

CCP4BB Home

CCP4BB Home

CCP4BB  June 2008

CCP4BB June 2008

Options

Subscribe or Unsubscribe

Subscribe or Unsubscribe

Log In

Log In

Get Password

Get Password

Subject:

Re: Activity of a mutant enzyme compared to wild type - puzzle

From:

Roger Rowlett <[log in to unmask]>

Reply-To:

Roger Rowlett <[log in to unmask]>

Date:

Thu, 12 Jun 2008 10:46:34 -0400

Content-Type:

text/plain

Parts/Attachments:

Parts/Attachments

text/plain (110 lines)

On a general note, it is not unusual at all for a random mutation (i.e., 
one not in the active or regulatory site of an enzyme, and not 
significantly connected with the catalytic or regulatory mechanism) to 
affect the rate constant of an enzyme-catalyzed reaction (kcat or 
kcat/Km) by a factor of 2 or more. Most enzyme kineticists don't get too 
excited about assigning an important role to a mutated residue unless 
one of the rate constants changes by a factor of 10. One danger in 
interpreting mutagenesis experiments is doing only a single kinetics 
measurement at a fixed pH and/or substrate concentration. If the 
mutation affects the pKa of enzyme catalytic groups or the effective Km 
of the substrate, a difference in rate will be noted, even though the 
fundamental rate constants kcat or kcat/Km, or their pH-independent 
maximal values, may not have significantly changed. Just my $0.02.

-- 
------------------------------------------------------------------------
Roger S. Rowlett
Professor
Colgate University Presidential Scholar
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: [log in to unmask]

Mischa Machius wrote:
> I assume you are talking about a sugar-binding enzyme ;) I have some
> aspects to consider in addition to what Artem raises. Many effects of
> a mutation are not recognizable in a static crystal structure or even
> in an NMR structure. For example, it is usually difficult to assess
> the thermodynamics of substrate binding, not to mention the kinetics.
> Multi-valent substrates usually display some sort of cooperativity for
> the binding process, which you might have affected by mutating one of
> the subsites. You might be able to obtain some hints from a Michaelis-
> Menten analysis of the mutant compared to the wild type, but that
> would only be a start. Your crystallographic result of a less occupied
> substrate-binding site for the mutant serves as a hint as well, but
> such results are hardly conclusive. You will have to follow up with
> more rigorous methods, such as ITC (thermodynamics of binding) and
> time-resolved methods (kinetics of binding).
>
> One example of an effect of a mutation that is usually not
> recognizable in a crystal structure has to do with substrate guiding.
> In this case, the mutation has changed the surface of the protein,
> thus affecting how well the multi-valent substrate can approach and
> wiggle itself into the binding site. Once in the binding site, it is
> structurally virtually indistinguishable from the wild-type.
>
> Ah, the nightmares of interpreting crystal structures in terms of
> biology!
>
> Good luck! Best - MM
>
>
> On Jun 11, 2008, at 7:21 PM, Narayanan Ramasubbu wrote:
>
>   
>> Dear all:
>> I have a single residue mutant whose enzyme activity is about 50% of
>> the wild type. Interestingly, the mutation
>> is in a region that involves a secondary site but not the active
>> site. The two structures with or without ligands
>> fit well (0.18 A) and the metal binding and cofactor binding sites
>> are all preserved in the mutant. The one difference
>> noticed is that the ligand does not fill the active site (partially
>> occupied subsites) unlike the wild type where all the
>> subsites are occupied. Water structure around the actives site
>> residues are "identical".
>>
>> I looked at the electrostatics and both surfaces look similar (not
>> an expert).
>>
>> There are some residues whose sides chains show some positional
>> disorder and these residues are at the edges of the
>> active site.
>>
>> The resolution of the both data sets are 1.5A.
>> The mutant enzyme was derived by MR.
>>
>> One another possibility that I want to look at is to compare the
>> compactness of the two enzyme structures.
>> What is the best way to compare that? I am wondering whether the
>> "breathing" that was mentioned for some enzymes
>> might be playing a role in the mutant enzyme.
>>
>> Also, I would appreciate comments on other possible explanations for
>> this unusual (?) behavior.
>>
>>
>> Thanks a lot
>>
>> Subbu
>>     
>
>
> --------------------------------------------------------------------------------
> Mischa Machius, PhD
> Associate Professor
> Department of Biochemistry
> UT Southwestern Medical Center at Dallas
> 5323 Harry Hines Blvd.; ND10.214A
> Dallas, TX 75390-8816; U.S.A.
> Tel: +1 214 645 6381
> Fax: +1 214 645 6353
>   

Top of Message | Previous Page | Permalink

JiscMail Tools


RSS Feeds and Sharing


Advanced Options


Archives

March 2024
February 2024
January 2024
December 2023
November 2023
October 2023
September 2023
August 2023
July 2023
June 2023
May 2023
April 2023
March 2023
February 2023
January 2023
December 2022
November 2022
October 2022
September 2022
August 2022
July 2022
June 2022
May 2022
April 2022
March 2022
February 2022
January 2022
December 2021
November 2021
October 2021
September 2021
August 2021
July 2021
June 2021
May 2021
April 2021
March 2021
February 2021
January 2021
December 2020
November 2020
October 2020
September 2020
August 2020
July 2020
June 2020
May 2020
April 2020
March 2020
February 2020
January 2020
December 2019
November 2019
October 2019
September 2019
August 2019
July 2019
June 2019
May 2019
April 2019
March 2019
February 2019
January 2019
December 2018
November 2018
October 2018
September 2018
August 2018
July 2018
June 2018
May 2018
April 2018
March 2018
February 2018
January 2018
December 2017
November 2017
October 2017
September 2017
August 2017
July 2017
June 2017
May 2017
April 2017
March 2017
February 2017
January 2017
December 2016
November 2016
October 2016
September 2016
August 2016
July 2016
June 2016
May 2016
April 2016
March 2016
February 2016
January 2016
December 2015
November 2015
October 2015
September 2015
August 2015
July 2015
June 2015
May 2015
April 2015
March 2015
February 2015
January 2015
December 2014
November 2014
October 2014
September 2014
August 2014
July 2014
June 2014
May 2014
April 2014
March 2014
February 2014
January 2014
December 2013
November 2013
October 2013
September 2013
August 2013
July 2013
June 2013
May 2013
April 2013
March 2013
February 2013
January 2013
December 2012
November 2012
October 2012
September 2012
August 2012
July 2012
June 2012
May 2012
April 2012
March 2012
February 2012
January 2012
December 2011
November 2011
October 2011
September 2011
August 2011
July 2011
June 2011
May 2011
April 2011
March 2011
February 2011
January 2011
December 2010
November 2010
October 2010
September 2010
August 2010
July 2010
June 2010
May 2010
April 2010
March 2010
February 2010
January 2010
December 2009
November 2009
October 2009
September 2009
August 2009
July 2009
June 2009
May 2009
April 2009
March 2009
February 2009
January 2009
December 2008
November 2008
October 2008
September 2008
August 2008
July 2008
June 2008
May 2008
April 2008
March 2008
February 2008
January 2008
December 2007
November 2007
October 2007
September 2007
August 2007
July 2007
June 2007
May 2007
April 2007
March 2007
February 2007
January 2007


JiscMail is a Jisc service.

View our service policies at https://www.jiscmail.ac.uk/policyandsecurity/ and Jisc's privacy policy at https://www.jisc.ac.uk/website/privacy-notice

For help and support help@jisc.ac.uk

Secured by F-Secure Anti-Virus CataList Email List Search Powered by the LISTSERV Email List Manager