1mcz
From Proteopedia
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'''BENZOYLFORMATE DECARBOXYLASE FROM PSEUDOMONAS PUTIDA COMPLEXED WITH AN INHIBITOR, R-MANDELATE''' | '''BENZOYLFORMATE DECARBOXYLASE FROM PSEUDOMONAS PUTIDA COMPLEXED WITH AN INHIBITOR, R-MANDELATE''' | ||
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[[Category: Hasson, M S.]] | [[Category: Hasson, M S.]] | ||
[[Category: Polovnikova, E S.]] | [[Category: Polovnikova, E S.]] | ||
| - | [[Category: | + | [[Category: Decarboxylase]] |
| - | [[Category: | + | [[Category: R-mandelate]] |
| - | [[Category: | + | [[Category: Thiamin diphosphate]] |
| - | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sat May 3 00:53:55 2008'' | |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | |
Revision as of 21:53, 2 May 2008
BENZOYLFORMATE DECARBOXYLASE FROM PSEUDOMONAS PUTIDA COMPLEXED WITH AN INHIBITOR, R-MANDELATE
Overview
Benzoylformate decarboxylase is a member of the family of enzymes that are dependent on the cofactor thiamin diphosphate. A structure of this enzyme binding (R)-mandelate, a competitive inhibitor, suggests that at least two hydrogen bonds are formed between the substrate, benzoylformate, and active site side chains. The first is between the carboxylate group of benzoylformate and the hydroxyl group of S26, and the second is between carbonyl group of the substrate and an imidazole nitrogen of H70. Steady-state kinetic studies indicate that the catalytic parameters are strongly affected in three active site mutants, S26A, H70A, and H281A. The K(m) of S26A was increased most dramatically, 25-fold more than that of the wild-type enzyme, while the K(i) of (R)-mandelate was increased 100-fold, suggesting that the serine hydroxyl is important for substrate binding. The k(cat) of H70A is reduced more than 3 orders of magnitude, strongly implicating this residue in catalysis, and H281 showed significant, but smaller magnitude, effects on both K(m) and k(cat). Stopped-flow experiments using an alternative substrate, p-nitrobenzoylformate, lead to kinetic resolution of the fate of key thiamin diphosphate-bound intermediates. Together, the experimental results suggest the following roles for residues in the active site. The residue H70 is important for the protonation of the 2-alpha-mandelyl-ThDP intermediate, thereby assisting in decarboxylation, and for the deprotonation of the 2-alpha-hydroxybenzyl-ThDP intermediate, aiding product release. H281 is involved in protonation of the enamine. Surprisingly, S26 appears to be involved not only in substrate binding but also in other steps of the reaction.
About this Structure
1MCZ is a Single protein structure of sequence from Pseudomonas putida. Full crystallographic information is available from OCA.
Reference
Structural and kinetic analysis of catalysis by a thiamin diphosphate-dependent enzyme, benzoylformate decarboxylase., Polovnikova ES, McLeish MJ, Sergienko EA, Burgner JT, Anderson NL, Bera AK, Jordan F, Kenyon GL, Hasson MS, Biochemistry. 2003 Feb 25;42(7):1820-30. PMID:12590569 Page seeded by OCA on Sat May 3 00:53:55 2008
