1cla
From Proteopedia
(New page: 200px<br /><applet load="1cla" size="450" color="white" frame="true" align="right" spinBox="true" caption="1cla, resolution 2.34Å" /> '''EVIDENCE FOR TRANSIT...) |
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| - | [[Image:1cla.jpg|left|200px]]<br /><applet load="1cla" size=" | + | [[Image:1cla.jpg|left|200px]]<br /><applet load="1cla" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1cla, resolution 2.34Å" /> | caption="1cla, resolution 2.34Å" /> | ||
'''EVIDENCE FOR TRANSITION-STATE STABILIZATION BY SERINE-148 IN THE CATALYTIC MECHANISM OF CHLORAMPHENICOL ACETYLTRANSFERASE'''<br /> | '''EVIDENCE FOR TRANSITION-STATE STABILIZATION BY SERINE-148 IN THE CATALYTIC MECHANISM OF CHLORAMPHENICOL ACETYLTRANSFERASE'''<br /> | ||
==Overview== | ==Overview== | ||
| - | The function of conserved Ser-148 of chloramphenicol acetyltransferase | + | The function of conserved Ser-148 of chloramphenicol acetyltransferase (CAT) has been investigated by site-directed mutagenesis. Modeling studies (P. C. E. Moody and A. G. W. Leslie, unpublished results) suggested that the hydroxyl group of Ser-148 could be involved in transition-state stabilization via a hydrogen bond to the oxyanion of the putative tetrahedral intermediate. Replacement of serine by alanine results in a mutant enzyme (Ala-148 CAT) with kcat reduced 53-fold and only minor changes in Km values for chloramphenicol and acetyl-CoA. The Ser-148----Gly substitution gives rise to a mutant enzyme (Gly-148 CAT) with kcat reduced only 10-fold. A water molecule may partially replace the hydrogen-bonding potential of Ser-148 in Gly-148 CAT. The three-dimensional structure of Ala-148 CAT at 2.34-A resolution is isosteric with that of wild-type CAT with two exceptions: the absence of the Ser-148 hydroxyl group and the loss of one poorly ordered water molecule from the active site region. The results are consistent with a catalytic role for Ser-148 rather than a structural one and support the hypothesis that Ser-148 is involved in transition-state stabilization. Ser-148 has also been replaced with cysteine and asparagine; the Ser-148----Cys mutation results in a 705-fold decrease in kcat and the Ser-148----Asn substitution in a 214-fold reduction in kcat. Removing the hydrogen bond donor (Ser-148----Ala or Gly) is less deleterious than replacing Ser-148 with alternative possible hydrogen bond donors (Ser-148----Cys or Asn). |
==About this Structure== | ==About this Structure== | ||
| - | 1CLA is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with CO and CLM as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Chloramphenicol_O-acetyltransferase Chloramphenicol O-acetyltransferase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.3.1.28 2.3.1.28] Full crystallographic information is available from [http:// | + | 1CLA is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=CO:'>CO</scene> and <scene name='pdbligand=CLM:'>CLM</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Chloramphenicol_O-acetyltransferase Chloramphenicol O-acetyltransferase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.3.1.28 2.3.1.28] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1CLA OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Escherichia coli]] | [[Category: Escherichia coli]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Gibbs, M | + | [[Category: Gibbs, M R.]] |
| - | [[Category: Leslie, A | + | [[Category: Leslie, A G.W.]] |
[[Category: CLM]] | [[Category: CLM]] | ||
[[Category: CO]] | [[Category: CO]] | ||
[[Category: transferase (acyltransferase)]] | [[Category: transferase (acyltransferase)]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:07:12 2008'' |
Revision as of 10:07, 21 February 2008
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EVIDENCE FOR TRANSITION-STATE STABILIZATION BY SERINE-148 IN THE CATALYTIC MECHANISM OF CHLORAMPHENICOL ACETYLTRANSFERASE
Overview
The function of conserved Ser-148 of chloramphenicol acetyltransferase (CAT) has been investigated by site-directed mutagenesis. Modeling studies (P. C. E. Moody and A. G. W. Leslie, unpublished results) suggested that the hydroxyl group of Ser-148 could be involved in transition-state stabilization via a hydrogen bond to the oxyanion of the putative tetrahedral intermediate. Replacement of serine by alanine results in a mutant enzyme (Ala-148 CAT) with kcat reduced 53-fold and only minor changes in Km values for chloramphenicol and acetyl-CoA. The Ser-148----Gly substitution gives rise to a mutant enzyme (Gly-148 CAT) with kcat reduced only 10-fold. A water molecule may partially replace the hydrogen-bonding potential of Ser-148 in Gly-148 CAT. The three-dimensional structure of Ala-148 CAT at 2.34-A resolution is isosteric with that of wild-type CAT with two exceptions: the absence of the Ser-148 hydroxyl group and the loss of one poorly ordered water molecule from the active site region. The results are consistent with a catalytic role for Ser-148 rather than a structural one and support the hypothesis that Ser-148 is involved in transition-state stabilization. Ser-148 has also been replaced with cysteine and asparagine; the Ser-148----Cys mutation results in a 705-fold decrease in kcat and the Ser-148----Asn substitution in a 214-fold reduction in kcat. Removing the hydrogen bond donor (Ser-148----Ala or Gly) is less deleterious than replacing Ser-148 with alternative possible hydrogen bond donors (Ser-148----Cys or Asn).
About this Structure
1CLA is a Single protein structure of sequence from Escherichia coli with and as ligands. Active as Chloramphenicol O-acetyltransferase, with EC number 2.3.1.28 Full crystallographic information is available from OCA.
Reference
Evidence for transition-state stabilization by serine-148 in the catalytic mechanism of chloramphenicol acetyltransferase., Lewendon A, Murray IA, Shaw WV, Gibbs MR, Leslie AG, Biochemistry. 1990 Feb 27;29(8):2075-80. PMID:2109633
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