Orotidine-5'-monophosphate decarboxylase catalysis: Kinetic isotope effects and the state of hybridization of a bound transition-state analogue
- Univ. of North Carolina School of Medicine, Chapel Hill (USA)
The enzymatic decarboxylation of orotidine 5'-monophosphate may proceed by an addition-elimination mechanism involving a covalently bound intermediate or by elimination of CO2 to generate a nitrogen ylide. In an attempt to distinguish between these two alternatives, 1-(phosphoribosyl)barbituric acid was synthesized with 13C at the 5-position. Interaction of this potential transition-state analogue inhibitor with yeast orotidine-5'-monophosphate decarboxylase resulted in a small (0.6 ppm) downfield displacement of the C-5 resonance, indicating no rehybridization of the kind that might have been expected to accompany 5,6-addition of an enzyme nucleophile. When the substrate orotidine 5'-monophosphate was synthesized with deuterium at C-5, no significant change in kcat (H/D = 0.99 +/- 0.06) or kcat/KM (H/D = 1.00 +/- 0.06) was found to result, suggesting that C-5 does not undergo significant changes in geometry before or during the step that determines the rate of the catalytic process. These results are consistent with a nitrogen ylide mechanism and offer no support for the intervention of covalently bound intermediates in the catalytic process.
- OSTI ID:
- 6810086
- Journal Information:
- Biochemistry; (USA), Vol. 29:13; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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Mechanism of the Orotidine 5′-Monophosphate Decarboxylase-Catalyzed Reaction: Evidence for Substrate Destabilization
Investigation of the enzymatic mechanism of yeast orotidine-5'-monophosphate decarboxylase using sup 13 C kinetic isotope effects
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DECARBOXYLASES
CATALYSIS
DEUTERIUM
ISOTOPE EFFECTS
CARBON 13
DECARBOXYLATION
NUCLEAR MAGNETIC RESONANCE
PROTEIN STRUCTURE
SUBSTRATES
TRACER TECHNIQUES
CARBON ISOTOPES
CARBON-CARBON LYASES
CARBOXY-LYASES
CHEMICAL REACTIONS
ENZYMES
EVEN-ODD NUCLEI
HYDROGEN ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
LYASES
MAGNETIC RESONANCE
NUCLEI
ODD-ODD NUCLEI
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STABLE ISOTOPES
550201* - Biochemistry- Tracer Techniques