Mechanism of the orotidine 5′-monophosphate decarboxylase-catalyzed reaction

Effect of solvent viscosity on kinetic constants

B. Mc Kay Wood, Kui K. Chan, Tina L. Amyes, John P. Richard, John Alan Gerlt

Research output: Contribution to journalArticle

Abstract

Orotidine 5′-monophosphate decarboxylase (OMPDC) is an exceptionally proficient catalyst: the rate acceleration (kcat/knon) is 7.1 × 1016, and the proficiency [(kcat/K M)/knon] is 4.8 × 1022M-1. The structural basis for the large rate acceleration and proficiency is unknown, although the mechanism has been established to involve a stabilized carbanion intermediate. To provide reaction coordinate context for interpretation of the values of kcat, kcat/KM, and kinetic isotope effects, we investigated the effect of solvent viscosity on kcat and kcat/KM for the OMPDCs from Methanothermobacter thermautotrophicus (MtOMPDC) and Saccharomyces cerevisiae (ScOMPDC). For MtOMPDC, we used not only the natural OMP substrate but also a catalytically impaired mutant (D70N) and a more reactive substrate (FOMP); for ScOMPDC, we used OMP and FOMP. With MtOMPDC and OMP, kcat is independent of solvent viscosity, indicating that decarboxylation is fully rate-determining; kcat/KM displays a fractional dependence of solvent viscosity, suggesting that both substrate binding and decarboxylation determine this kinetic constant. For ScOMPDC with OMP, we observed that both k cat and kcat/KM are fractionally dependent on solvent viscosity, suggesting that the rates of substrate binding, decarboxylation, and product dissociation are similar. Consistent with these interpretations, for both enzymes with FOMP, the increases in the values of kcat and kcat/KM are much less than expected based on the ability of the 5-fluoro substituent to stabilize the anionic intermediate; i.e., substrate binding and product dissociation mask the kinetic effects of stabilization of the intermediate by the substituent.

Original languageEnglish (US)
Pages (from-to)5510-5517
Number of pages8
JournalBiochemistry
Volume48
Issue number24
DOIs
StatePublished - Jun 23 2009

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Carboxy-Lyases
Viscosity
Decarboxylation
Kinetics
Substrates
Methanobacteriaceae
Masks
Isotopes
Saccharomyces cerevisiae
Cats
Yeast
Stabilization
orotidylic acid
Enzymes
Catalysts

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mechanism of the orotidine 5′-monophosphate decarboxylase-catalyzed reaction : Effect of solvent viscosity on kinetic constants. / Wood, B. Mc Kay; Chan, Kui K.; Amyes, Tina L.; Richard, John P.; Gerlt, John Alan.

In: Biochemistry, Vol. 48, No. 24, 23.06.2009, p. 5510-5517.

Research output: Contribution to journalArticle

Wood, B. Mc Kay ; Chan, Kui K. ; Amyes, Tina L. ; Richard, John P. ; Gerlt, John Alan. / Mechanism of the orotidine 5′-monophosphate decarboxylase-catalyzed reaction : Effect of solvent viscosity on kinetic constants. In: Biochemistry. 2009 ; Vol. 48, No. 24. pp. 5510-5517.
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abstract = "Orotidine 5′-monophosphate decarboxylase (OMPDC) is an exceptionally proficient catalyst: the rate acceleration (kcat/knon) is 7.1 × 1016, and the proficiency [(kcat/K M)/knon] is 4.8 × 1022M-1. The structural basis for the large rate acceleration and proficiency is unknown, although the mechanism has been established to involve a stabilized carbanion intermediate. To provide reaction coordinate context for interpretation of the values of kcat, kcat/KM, and kinetic isotope effects, we investigated the effect of solvent viscosity on kcat and kcat/KM for the OMPDCs from Methanothermobacter thermautotrophicus (MtOMPDC) and Saccharomyces cerevisiae (ScOMPDC). For MtOMPDC, we used not only the natural OMP substrate but also a catalytically impaired mutant (D70N) and a more reactive substrate (FOMP); for ScOMPDC, we used OMP and FOMP. With MtOMPDC and OMP, kcat is independent of solvent viscosity, indicating that decarboxylation is fully rate-determining; kcat/KM displays a fractional dependence of solvent viscosity, suggesting that both substrate binding and decarboxylation determine this kinetic constant. For ScOMPDC with OMP, we observed that both k cat and kcat/KM are fractionally dependent on solvent viscosity, suggesting that the rates of substrate binding, decarboxylation, and product dissociation are similar. Consistent with these interpretations, for both enzymes with FOMP, the increases in the values of kcat and kcat/KM are much less than expected based on the ability of the 5-fluoro substituent to stabilize the anionic intermediate; i.e., substrate binding and product dissociation mask the kinetic effects of stabilization of the intermediate by the substituent.",
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AU - Gerlt, John Alan

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N2 - Orotidine 5′-monophosphate decarboxylase (OMPDC) is an exceptionally proficient catalyst: the rate acceleration (kcat/knon) is 7.1 × 1016, and the proficiency [(kcat/K M)/knon] is 4.8 × 1022M-1. The structural basis for the large rate acceleration and proficiency is unknown, although the mechanism has been established to involve a stabilized carbanion intermediate. To provide reaction coordinate context for interpretation of the values of kcat, kcat/KM, and kinetic isotope effects, we investigated the effect of solvent viscosity on kcat and kcat/KM for the OMPDCs from Methanothermobacter thermautotrophicus (MtOMPDC) and Saccharomyces cerevisiae (ScOMPDC). For MtOMPDC, we used not only the natural OMP substrate but also a catalytically impaired mutant (D70N) and a more reactive substrate (FOMP); for ScOMPDC, we used OMP and FOMP. With MtOMPDC and OMP, kcat is independent of solvent viscosity, indicating that decarboxylation is fully rate-determining; kcat/KM displays a fractional dependence of solvent viscosity, suggesting that both substrate binding and decarboxylation determine this kinetic constant. For ScOMPDC with OMP, we observed that both k cat and kcat/KM are fractionally dependent on solvent viscosity, suggesting that the rates of substrate binding, decarboxylation, and product dissociation are similar. Consistent with these interpretations, for both enzymes with FOMP, the increases in the values of kcat and kcat/KM are much less than expected based on the ability of the 5-fluoro substituent to stabilize the anionic intermediate; i.e., substrate binding and product dissociation mask the kinetic effects of stabilization of the intermediate by the substituent.

AB - Orotidine 5′-monophosphate decarboxylase (OMPDC) is an exceptionally proficient catalyst: the rate acceleration (kcat/knon) is 7.1 × 1016, and the proficiency [(kcat/K M)/knon] is 4.8 × 1022M-1. The structural basis for the large rate acceleration and proficiency is unknown, although the mechanism has been established to involve a stabilized carbanion intermediate. To provide reaction coordinate context for interpretation of the values of kcat, kcat/KM, and kinetic isotope effects, we investigated the effect of solvent viscosity on kcat and kcat/KM for the OMPDCs from Methanothermobacter thermautotrophicus (MtOMPDC) and Saccharomyces cerevisiae (ScOMPDC). For MtOMPDC, we used not only the natural OMP substrate but also a catalytically impaired mutant (D70N) and a more reactive substrate (FOMP); for ScOMPDC, we used OMP and FOMP. With MtOMPDC and OMP, kcat is independent of solvent viscosity, indicating that decarboxylation is fully rate-determining; kcat/KM displays a fractional dependence of solvent viscosity, suggesting that both substrate binding and decarboxylation determine this kinetic constant. For ScOMPDC with OMP, we observed that both k cat and kcat/KM are fractionally dependent on solvent viscosity, suggesting that the rates of substrate binding, decarboxylation, and product dissociation are similar. Consistent with these interpretations, for both enzymes with FOMP, the increases in the values of kcat and kcat/KM are much less than expected based on the ability of the 5-fluoro substituent to stabilize the anionic intermediate; i.e., substrate binding and product dissociation mask the kinetic effects of stabilization of the intermediate by the substituent.

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