Orotidine-5'-monophosphate decarboxylase catalysis: Kinetic isotope effects and the state of hybridization of a bound transition-state analogue

Journal Article · Tue Apr 03 00:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00465a007· OSTI ID:6810086

Acheson, S A; Bell, J B; Jones, M E; Wolfenden, R ^[1]

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1. 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

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
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
RESONANCE
STABLE ISOTOPES
550201* - Biochemistry- Tracer Techniques