General acid/base - Revision history

Spencer Williams at 10:38, 4 August 2013

2013-08-04T10:38:37Z

Spencer Williams at 12:02, 30 April 2013

2013-04-30T12:02:34Z

Harry Brumer at 18:02, 19 February 2010

2010-02-19T18:02:10Z

Spencer Williams at 09:23, 22 January 2010

2010-01-22T09:23:21Z

Spencer Williams at 06:35, 6 September 2009

2009-09-06T06:35:17Z

Spencer Williams at 06:27, 6 September 2009

2009-09-06T06:27:14Z

Spencer Williams at 02:01, 1 September 2009

2009-09-01T02:01:34Z

Spencer Williams: Created page with ' * Author: Spencer Williams * Responsible Curator: Spencer Williams ---- == Overview == The term '''catalytic acid/base''' re…'

2009-09-01T01:58:03Z

Created page with ' * Author: Spencer Williams * Responsible Curator: Spencer Williams ---- == Overview == The term '''catalytic acid/base''' re…'

New page

* Author: [[User:SpencerWilliams|Spencer Williams]]
* Responsible Curator: [[User:SpencerWilliams|Spencer Williams]]
----
== Overview ==

The term '''catalytic acid/base''' refers to an amino acid residue in a [[glycoside hydrolase]] or a related enzyme that participates in the mechanism of hydrolysis by removing or adding a proton. The mechanism may be a [[retaining]] or [[inverting]] mechanism.

[[Image:Retaining_glycosidase_mechanism_1.png|centre]]

==Methods for indentifying the catalytic acid/base==

== References ==
<biblio>

</biblio>
[[Category:Definitions and explanations]]

@@ Line 14: / Line 14: @@
 ===Kinetic analysis of mutants===
-Assignment of the acid/base residues may be achieved by the kinetic analysis of enzyme mutants in which the candidate residues have been replaced by a non-nucleophilic residue. Alanine is preferred as a replacement residue owing to its small size. Glycine is non-optimal, as the lack of an alpha-substituent may lead to differences in conformation. Alanine mutants typically have ''k''<sub>cat</sub> values 1,000-fold lower than the wildtype enzyme when assessed against the natural substrate <cite>1</cite>.This effect is much smaller when the substrate possesses a good leaving group, such as a fluoride or a 2,4-dinitrophenyl group, as in these cases the leaving groups do not require general acid catalysis. In these cases the ''k''<sub>cat</sub> values are unaffected, but as the deglycosylation step requires general base catalysis the [[intermediate]] accumulates, leading to a reduction in the ''K''<sub>M</sub> value. A further test for the identity of the acid base residue is the observation of a 'chemical rescue' effect. In the presence of a small anionic nucleophilic group such as azide, fluoride or formate, the glycosyl enzyme will be turned over. In this case the product of 'chemical rescue' should be a glycosyl azide, fluoride or formate with anomeric stereochemistry matching that of the starting glycoside.
+Assignment of the acid/base residues may be achieved by the kinetic analysis of enzyme mutants in which the candidate residues have been replaced by a non-nucleophilic residue. Alanine is preferred as a replacement residue owing to its small size. Glycine is non-optimal, as the lack of an alpha-substituent may lead to differences in conformation. Alanine mutants typically have ''k''<sub>cat</sub> values 1,000-fold lower than the wildtype enzyme when assessed against the natural substrate <cite>Ly1999</cite>.This effect is much smaller when the substrate possesses a good leaving group, such as a fluoride or a 2,4-dinitrophenyl group, as in these cases the leaving groups do not require general acid catalysis. In these cases the ''k''<sub>cat</sub> values are unaffected, but as the deglycosylation step requires general base catalysis the [[intermediate]] accumulates, leading to a reduction in the ''K''<sub>M</sub> value. A further test for the identity of the acid base residue is the observation of a 'chemical rescue' effect. In the presence of a small anionic nucleophilic group such as azide, fluoride or formate, the glycosyl enzyme will be turned over. In this case the product of 'chemical rescue' should be a glycosyl azide, fluoride or formate with anomeric stereochemistry matching that of the starting glycoside.
 ===Mechanism-based labelling===
@@ Line 22: / Line 22: @@
 == References ==
 <biblio>
-#1 pmid=10872458
+#Ly1999 pmid=10872458
 </biblio>
 [[Category:Definitions and explanations]]

@@ Line 8: / Line 8: @@
 The term '''general acid/base''' (also catalytic acid/base) refers to an amino acid residue in a [[glycoside hydrolase]] or a related enzyme that participates in the mechanism of hydrolysis by removing or adding a proton (or both). The mechanism may be a [[retaining]] or [[inverting]] mechanism. General acid/base catalysis differs from specific acid/base catalysis as in the latter it is the solvent that acts as the acid or base.
-[[Image:General_acid_base.png|centre]]
+[[Image:General_acid_base.png|centre|800px]]
 ==Methods for indentifying the general acid/base==

← Older revision		Revision as of 18:02, 19 February 2010
Line 1:		Line 1:
−
−
	{{CuratorApproved}}		{{CuratorApproved}}

← Older revision		Revision as of 09:23, 22 January 2010
Line 1:		Line 1:
		+
		+
		+	{{CuratorApproved}}

	* Author: [[User:SpencerWilliams\|Spencer Williams]]		* Author: [[User:SpencerWilliams\|Spencer Williams]]

@@ Line 1: / Line 1: @@
 * Author: [[User:SpencerWilliams|Spencer Williams]]
@@ Line 8: / Line 7: @@
 The term '''general acid/base''' (also catalytic acid/base) refers to an amino acid residue in a [[glycoside hydrolase]] or a related enzyme that participates in the mechanism of hydrolysis by removing or adding a proton (or both). The mechanism may be a [[retaining]] or [[inverting]] mechanism. General acid/base catalysis differs from specific acid/base catalysis as in the latter it is the solvent that acts as the acid or base.
-[[Image:Retaining_glycosidase_mechanism_1.png|centre]]
+[[Image:General_acid_base.png|centre]]
 ==Methods for indentifying the general acid/base==