+ Site Statistics
References:
54,258,434
Abstracts:
29,560,870
PMIDs:
28,072,757
+ Search Articles
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ PDF Full Text
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Translate
+ Recently Requested

Proton transfer from C-6 of uridine 5'-monophosphate catalyzed by orotidine 5'-monophosphate decarboxylase: formation and stability of a vinyl carbanion intermediate and the effect of a 5-fluoro substituent



Proton transfer from C-6 of uridine 5'-monophosphate catalyzed by orotidine 5'-monophosphate decarboxylase: formation and stability of a vinyl carbanion intermediate and the effect of a 5-fluoro substituent



Journal of the American Chemical Society 134(35): 14580-14594



The exchange for deuterium of the C-6 protons of uridine 5'-monophosphate (UMP) and 5-fluorouridine 5'-monophosphate (F-UMP) catalyzed by yeast orotidine 5'-monophosphate decarboxylase (ScOMPDC) at pD 6.5-9.3 and 25 °C was monitored by (1)H NMR spectroscopy. Deuterium exchange proceeds by proton transfer from C-6 of the bound nucleotide to the deprotonated side chain of Lys-93 to give the enzyme-bound vinyl carbanion. The pD-rate profiles for k(cat) give turnover numbers for deuterium exchange into enzyme-bound UMP and F-UMP of 1.2 × 10(-5) and 0.041 s(-1), respectively, so that the 5-fluoro substituent results in a 3400-fold increase in the first-order rate constant for deuterium exchange. The binding of UMP and F-UMP to ScOMPDC results in 0.5 and 1.4 unit decreases, respectively, in the pK(a) of the side chain of the catalytic base Lys-93, showing that these nucleotides bind preferentially to the deprotonated enzyme. We also report the first carbon acid pK(a) values for proton transfer from C-6 of uridine (pK(CH) = 28.8) and 5-fluorouridine (pK(CH) = 25.1) in aqueous solution. The stabilizing effects of the 5-fluoro substituent on C-6 carbanion formation in solution (5 kcal/mol) and at ScOMPDC (6 kcal/mol) are similar. The binding of UMP and F-UMP to ScOMPDC results in a greater than 5 × 10(9)-fold increase in the equilibrium constant for proton transfer from C-6, so that ScOMPDC stabilizes the bound vinyl carbanions, relative to the bound nucleotides, by at least 13 kcal/mol. The pD-rate profile for k(cat)/K(m) for deuterium exchange into F-UMP gives the intrinsic second-order rate constant for exchange catalyzed by the deprotonated enzyme as 2300 M(-1) s(-1). This was used to calculate a total rate acceleration for ScOMPDC-catalyzed deuterium exchange of 3 × 10(10) M(-1), which corresponds to a transition-state stabilization for deuterium exchange of 14 kcal/mol. We conclude that a large portion of the total transition-state stabilization for the decarboxylation of orotidine 5'-monophosphate can be accounted for by stabilization of the enzyme-bound vinyl carbanion intermediate of the stepwise reaction.

(PDF emailed within 0-6 h: $19.90)

Accession: 055260575

Download citation: RISBibTeXText

PMID: 22812629

DOI: 10.1021/ja3058474


Related references

Formation and stability of a vinyl carbanion at the active site of orotidine 5'-monophosphate decarboxylase: pKa of the C-6 proton of enzyme-bound UMP. Journal of the American Chemical Society 130(5): 1574-1575, 2008

Product deuterium isotope effects for orotidine 5'-monophosphate decarboxylase: effect of changing substrate and enzyme structure on the partitioning of the vinyl carbanion reaction intermediate. Journal of the American Chemical Society 132(20): 7018-7024, 2010

Product deuterium isotope effect for orotidine 5'-monophosphate decarboxylase: evidence for the existence of a short-lived carbanion intermediate. Journal of the American Chemical Society 129(43): 12946-7, 2007

Stability of the 6-carbanion of uracil analogues: mechanistic implications for model reactions of orotidine-5'-monophosphate decarboxylase. Organic Letters 8(26): 6019-6022, 2006

Isolation and characterization of the orotidine 5'-monophosphate decarboxylase domain of the multifunctional protein uridine 5'-monophosphate synthase. Journal of Biological Chemistry 260(16): 9443-9451, 1985

A nonradioactive high-performance liquid chromatographic microassay for uridine 5'-monophosphate synthase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate decarboxylase. Analytical Biochemistry 299(2): 162-168, 2001

Role of a guanidinium cation-phosphodianion pair in stabilizing the vinyl carbanion intermediate of orotidine 5'-phosphate decarboxylase-catalyzed reactions. Biochemistry 52(42): 7500-7511, 2013

Chemical models and their mechanistic implications for the transformation of 6-cyanouridine 5'-monophosphate catalyzed by orotidine 5'-monophosphate decarboxylase. Chemical Communications 46(26): 4821-4823, 2010

Chemical models and their mechanistic implications for the transformation of 6-cyanouridine 5'-monophosphate catalyzed by orotidine 5'-monophosphate decarboxylase. Nucleic Acids Symposium Series: 297-298, 2008

Structural evidence for a 1,2-enediolate intermediate in the reaction catalyzed by 3-keto-L-gulonate 6-phosphate decarboxylase, a member of the orotidine 5'-monophosphate decarboxylase suprafamily. Biochemistry 42(42): 12133-12142, 2003

Catalysis by orotidine 5'-monophosphate decarboxylase: effect of 5-fluoro and 4'-substituents on the decarboxylation of two-part substrates. Biochemistry 52(3): 537-546, 2013

Carbon isotope effect study on orotidine 5'-monophosphate decarboxylase: support for an anionic intermediate. Biochemistry 47(2): 798-803, 2007

Mechanism of the orotidine 5'-monophosphate decarboxylase-catalyzed reaction: effect of solvent viscosity on kinetic constants. Biochemistry 48(24): 5510-5517, 2009

Structure-activity relationships of C6-uridine derivatives targeting plasmodia orotidine monophosphate decarboxylase. Journal of Medicinal Chemistry 51(3): 439-448, 2008

Establishing paradigms in the orotidine 5-monophosphate decarboxylase suprafamily from studies of the reaction catalyzed by 3-keto-L-gulonate 6-phosphate decarboxylase. Abstracts of Papers American Chemical Society 226(1-2): BIOL 116, 2003