1wcq
From Proteopedia
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==Overview== | ==Overview== | ||
| - | Mutants of the Micromonospora viridifaciens sialidase, Y370E and Y370F, are catalytically active retaining enzymes that operate by different | + | Mutants of the Micromonospora viridifaciens sialidase, Y370E and Y370F, are catalytically active retaining enzymes that operate by different mechanisms. Previous substitutions with smaller amino acids, including Y370D, yielded inverting sialidases. At least one water molecule can fit into the active-site cavity of this mutant and act as a nucleophile from the face opposite the leaving group (Biochemistry 2003, 42, 12 682). Thus, addition of a CH(2) unit (Asp versus Glu) changes the mechanism from inversion back to retention of configuration. Based on Bronsted beta(lg) values, it is proposed that the Y370E mutant reacts by a double-displacement mechanism (beta(lg) on k(cat)/K(m) -0.36+/-0.04) with Glu370 acting as the nucleophile. However, the Y370F mutant (beta(lg) on k(cat)/K(m) -0.79+/-0.12) reacts via a dissociative transition state. The crystal structure of the Y370F mutant complexed with 2-deoxy-2,3-dehydro-N-acetylneuraminic acid shows no significant active-site perturbation relative to the wild-type enzyme. |
==About this Structure== | ==About this Structure== | ||
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[[Category: Micromonospora viridifaciens]] | [[Category: Micromonospora viridifaciens]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Bennet, A | + | [[Category: Bennet, A J.]] |
[[Category: Newstead, S.]] | [[Category: Newstead, S.]] | ||
[[Category: Taylor, G.]] | [[Category: Taylor, G.]] | ||
| - | [[Category: Watson, J | + | [[Category: Watson, J N.]] |
[[Category: DAN]] | [[Category: DAN]] | ||
[[Category: GOL]] | [[Category: GOL]] | ||
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[[Category: sialidase]] | [[Category: sialidase]] | ||
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 15:42:51 2008'' |
Revision as of 13:42, 21 February 2008
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MUTAGENESIS OF THE NUCLEOPHILIC TYROSINE IN A BACTERIAL SIALIDASE TO PHENYLALANINE.
Overview
Mutants of the Micromonospora viridifaciens sialidase, Y370E and Y370F, are catalytically active retaining enzymes that operate by different mechanisms. Previous substitutions with smaller amino acids, including Y370D, yielded inverting sialidases. At least one water molecule can fit into the active-site cavity of this mutant and act as a nucleophile from the face opposite the leaving group (Biochemistry 2003, 42, 12 682). Thus, addition of a CH(2) unit (Asp versus Glu) changes the mechanism from inversion back to retention of configuration. Based on Bronsted beta(lg) values, it is proposed that the Y370E mutant reacts by a double-displacement mechanism (beta(lg) on k(cat)/K(m) -0.36+/-0.04) with Glu370 acting as the nucleophile. However, the Y370F mutant (beta(lg) on k(cat)/K(m) -0.79+/-0.12) reacts via a dissociative transition state. The crystal structure of the Y370F mutant complexed with 2-deoxy-2,3-dehydro-N-acetylneuraminic acid shows no significant active-site perturbation relative to the wild-type enzyme.
About this Structure
1WCQ is a Single protein structure of sequence from Micromonospora viridifaciens with , and as ligands. Active as Exo-alpha-sialidase, with EC number 3.2.1.18 Known structural/functional Site: . Full crystallographic information is available from OCA.
Reference
Two nucleophilic mutants of the Micromonospora viridifaciens sialidase operate with retention of configuration by two different mechanisms., Watson JN, Newstead S, Narine AA, Taylor G, Bennet AJ, Chembiochem. 2005 Nov;6(11):1999-2004. PMID:16206228
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