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1m03
From Proteopedia
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'''Mutant Streptomyces plicatus beta-hexosaminidase (D313A) in complex with product (GlcNAc)''' | '''Mutant Streptomyces plicatus beta-hexosaminidase (D313A) in complex with product (GlcNAc)''' | ||
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[[Category: Williams, S J.]] | [[Category: Williams, S J.]] | ||
[[Category: Withers, S G.]] | [[Category: Withers, S G.]] | ||
| - | [[Category: | + | [[Category: Beta-hexosaminidase]] |
| - | [[Category: | + | [[Category: Family 20 glycosidase]] |
| - | [[Category: | + | [[Category: Substrate assisted catalysis]] |
| - | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sat May 3 00:28:25 2008'' | |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | |
Revision as of 21:28, 2 May 2008
Mutant Streptomyces plicatus beta-hexosaminidase (D313A) in complex with product (GlcNAc)
Overview
SpHex, a retaining family 20 glycosidase from Streptomyces plicatus, catalyzes the hydrolysis of N-acetyl-beta-hexosaminides. Accumulating evidence suggests that the hydrolytic mechanism involves substrate-assisted catalysis wherein the 2-acetamido substituent acts as a nucleophile to form an oxazolinium ion intermediate. The role of a conserved aspartate residue (D313) in the active site of SpHex was investigated through kinetic and structural analyses of two variant enzymes, D313A and D313N. Three-dimensional structures of the wild-type and variant enzymes in product complexes with N-acetyl-d-glucosamine revealed substantial differences. In the D313A variant the 2-acetamido group was found in two conformations of which only one is able to aid in catalysis through anchimeric assistance. The mutation D313N results in a steric clash in the active site between Asn-313 and the 2-acetamido group preventing the 2-acetamido group from providing anchimeric assistance, consistent with the large reduction in catalytic efficiency and the insensitivity of this variant to chemical rescue. By comparison, the D313A mutation results in a shift in a shift in the pH optimum and a modest decrease in activity that can be rescued by using azide as an exogenous nucleophile. These structural and kinetic data provide evidence that Asp-313 stabilizes the transition states flanking the oxazoline intermediate and also assists to correctly orient the 2-acetamido group for catalysis. Based on analogous conserved residues in the family 18 chitinases and family 56 hyaluronidases, the roles played by the Asp-313 residue is likely general for all hexosaminidases using a mechanism involving substrate-assisted catalysis.
About this Structure
1M03 is a Single protein structure of sequence from Streptomyces plicatus. Full crystallographic information is available from OCA.
Reference
Aspartate 313 in the Streptomyces plicatus hexosaminidase plays a critical role in substrate-assisted catalysis by orienting the 2-acetamido group and stabilizing the transition state., Williams SJ, Mark BL, Vocadlo DJ, James MN, Withers SG, J Biol Chem. 2002 Oct 18;277(42):40055-65. Epub 2002 Aug 8. PMID:12171933 Page seeded by OCA on Sat May 3 00:28:25 2008
