Document Detail

Proton-nuclear magnetic resonance analyses of the substrate specificity of a beta-ketolase from Pseudomonas putida, acetopyruvate hydrolase.
MedLine Citation:
PMID:  10438778     Owner:  NLM     Status:  MEDLINE    
A revised purification of acetopyruvate hydrolase from orcinol-grown Pseudomonas putida ORC is described. This carbon-carbon bond hydrolase, which is the last inducible enzyme of the orcinol catabolic pathway, is monomeric with a molecular size of approximately 38 kDa; it hydrolyzes acetopyruvate to equimolar quantities of acetate and pyruvate. We have previously described the aqueous-solution structures of acetopyruvate at pH 7.5 and several synthesized analogues by (1)H-nuclear magnetic resonance (NMR)-Fourier transform (FT) experiments. Three (1)H signals (2.2 to 2.4 ppm) of the methyl group are assigned unambiguously to the carboxylate anions of 2,4-diketo, 2-enol-4-keto, and 2-hydrate-4-keto forms (40:50:10). A (1)H-NMR assay for acetopyruvate hydrolase was used to study the kinetics and stoichiometries of reactions within a single reaction mixture (0.7 ml) by monitoring the three methyl-group signals of acetopyruvate and of the products acetate and pyruvate. Examination of 4-tert-butyl-2,4-diketobutanoate hydrolysis by the same method allowed the conclusion that it is the carboxylate 2-enol form(s) or carbanion(s) that is the actual substrate(s) of hydrolysis. Substrate analogues of 2,4-diketobutanoate with 4-phenyl or 4-benzyl groups are very poor substrates for the enzyme, whereas the 4-cyclohexyl analogue is readily hydrolyzed. In aqueous solution, the arene analogues do not form a stable 2-enol structure but exist principally as a delocalized pi-electron system in conjugation with the aromatic ring. The effects of several divalent metal ions on solution structures were studied, and a tentative conclusion that the enol forms are coordinated to Mg(2+) bound to the enzyme was made. (1)H-(2)H exchange reactions showed the complete, fast equilibration of (2)H into the C-3 of acetopyruvate chemically; this accounts for the appearance of (2)H in the product pyruvate. The C-3 of the product pyruvate was similarly labelled, but this exchange was only enzyme catalyzed; the methyl group of acetate did not undergo an exchange reaction. The unexpected preference for bulky 4-alkyl-group analogues is discussed in an evolutionary context for carbon-carbon bond hydrolases. Routine one-dimensional (1)H-NMR in normal (1)H(2)O is a new method for rapid, noninvasive assays of enzymic activities to obtain the kinetics and stoichiometries of reactions in single reaction mixtures. Assessments of the solution structures of both substrates and products are also shown.
D Pokorny; L Brecker; M Pogorevc; W Steiner; H Griengl; T Kappe; D W Ribbons
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of bacteriology     Volume:  181     ISSN:  0021-9193     ISO Abbreviation:  J. Bacteriol.     Publication Date:  1999 Aug 
Date Detail:
Created Date:  1999-09-03     Completed Date:  1999-09-03     Revised Date:  2013-04-17    
Medline Journal Info:
Nlm Unique ID:  2985120R     Medline TA:  J Bacteriol     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  5051-9     Citation Subset:  IM    
Institute of Biotechnology, Technical University Graz, A-8010 Graz, Austria.
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MeSH Terms
Bacterial Proteins / analysis,  metabolism
Copper / pharmacology
Fourier Analysis
Hydrolases / analysis*,  metabolism*
Magnesium / pharmacology
Magnetic Resonance Spectroscopy
Manganese / pharmacology
Oxygenases / analysis,  metabolism
Pseudomonas putida / enzymology*
Pyruvates / metabolism*
Substrate Specificity
Water / chemistry
Reg. No./Substance:
0/Bacterial Proteins; 0/Protons; 0/Pyruvates; 5699-58-1/acetylpyruvic acid; 7439-95-4/Magnesium; 7439-96-5/Manganese; 7440-50-8/Copper; 7732-18-5/Water; EC 1.13.-/Oxygenases; EC 1.13.-/beta-carotene ketolase; EC 3.-/Hydrolases

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