Investigation of the enzymatic mechanism of yeast orotidine-5'-monophosphate decarboxylase using sup 13 C kinetic isotope effects

Journal Article · Tue Jun 25 00:00:00 EDT 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00239a020· OSTI ID:5032443

Smiley, J A; Bell, J B; Jones, M E ^[1]; Paneth, P; O'Leary, M H ^[2]

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1. Univ. of North Carolina, Chapel Hill (United States)
2. Univ. of Nebraska, Lincoln (United States)

Orotidine-5'-monophosphate decarboxylase (ODCase) from Saccharomyces cerevisiae displays an observed {sup 13}C kinetic isotope effect of 1.0247 {plus minus} 0.0008 at 25 C, pH 6.8. The observed isotope effect is sensitive to changes in the reaction medium, such as pH, temperature, or glycerol content. The value of 1.0494 {plus minus} 0.0006 measured at pH 4.0, 25 C, is not altered significantly by temperature or glycerol, and thus the intrinsic isotope effect for the reaction is apparently being observed under these conditions and decarboxylation is almost entirely rate-determining. These data require a catalytic mechanism with freely reversible binding and one in which a very limited contribution to the overall rate is made by chemical steps preceding decarboxylation; the zwitterion mechanism of Beak and Siegel, which involves only protonation of the pyrimidine ring, is such a mechanism. With use of an intrinsic isotope effect of 1.05, a partitioning factor of less than unity is calculated for ODCase at pH 6.0, 25 C. A quantitative kinetic analysis using this result excludes the possibility of an enzymatic mechanism involving covalent attachment of an enzyme nucleophile to C-5 of the pyrimidine ring. These data fit a kinetic model in which an enzyme proton necessary for catalysis is titrated at high pH, thus providing evidence for the catalytic mechanism of Beak and Siegal.

• OSTI ID: 5032443
• Journal Information: Biochemistry; (United States), Vol. 30:25; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
CARBON 13
ISOTOPE EFFECTS
DECARBOXYLASES
BIOCHEMICAL REACTION KINETICS
BIOCHEMISTRY
ENZYME ACTIVITY
NUCLEOTIDES
PH VALUE
SACCHAROMYCES CEREVISIAE
CARBON ISOTOPES
CARBON-CARBON LYASES
CARBOXY-LYASES
CHEMISTRY
ENZYMES
EUMYCOTA
EVEN-ODD NUCLEI
FUNGI
ISOTOPES
KINETICS
LIGHT NUCLEI
LYASES
MICROORGANISMS
NUCLEI
ORGANIC COMPOUNDS
PLANTS
REACTION KINETICS
SACCHAROMYCES
STABLE ISOTOPES
YEASTS
550201* - Biochemistry- Tracer Techniques