Document Detail


Structural and kinetic analysis of catalysis by a thiamin diphosphate-dependent enzyme, benzoylformate decarboxylase.
MedLine Citation:
PMID:  12590569     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Elena S Polovnikova; Michael J McLeish; Eduard A Sergienko; John T Burgner; Natalie L Anderson; Asim K Bera; Frank Jordan; George L Kenyon; Miriam S Hasson
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Biochemistry     Volume:  42     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  2003 Feb 
Date Detail:
Created Date:  2003-02-19     Completed Date:  2003-05-07     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1820-30     Citation Subset:  IM    
Affiliation:
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-1392, USA.
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MeSH Terms
Descriptor/Qualifier:
Alanine / genetics
Binding Sites / genetics
Binding, Competitive / genetics
Carboxy-Lyases / chemistry*,  genetics
Catalysis
Circular Dichroism
Crystallography, X-Ray
Enzyme Inhibitors / chemistry
Histidine / genetics
Kinetics
Ligands
Mandelic Acids / chemistry
Mutagenesis, Site-Directed
Pseudomonas putida / enzymology
Serine / genetics
Spectrophotometry
Thiamine Pyrophosphate / chemistry*
Grant Support
ID/Acronym/Agency:
GM-40570/GM/NIGMS NIH HHS; GM-50380/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Enzyme Inhibitors; 0/Ligands; 0/Mandelic Acids; 154-87-0/Thiamine Pyrophosphate; 56-41-7/Alanine; 56-45-1/Serine; 71-00-1/Histidine; 90-64-2/mandelic acid; EC 4.1.1.-/Carboxy-Lyases; EC 4.1.1.7/benzoylformate decarboxylase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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