Biology:Orotidine 5'-phosphate decarboxylase
| Orotidine-5'-phosphate decarboxylase | |||||||||
|---|---|---|---|---|---|---|---|---|---|
 E. coli OMP decarboxylase.[1] | |||||||||
| Identifiers | |||||||||
| EC number | 4.1.1.23 | ||||||||
| CAS number | 9024-62-8 | ||||||||
| Databases | |||||||||
| IntEnz | IntEnz view | ||||||||
| BRENDA | BRENDA entry | ||||||||
| ExPASy | NiceZyme view | ||||||||
| KEGG | KEGG entry | ||||||||
| MetaCyc | metabolic pathway | ||||||||
| PRIAM | profile | ||||||||
| PDB structures | RCSB PDB PDBe PDBsum | ||||||||
| Gene Ontology | AmiGO / QuickGO | ||||||||
| |||||||||
Orotidine 5'-phosphate decarboxylase (OMP decarboxylase) or orotidylate decarboxylase is an enzyme involved in pyrimidine biosynthesis. It catalyzes the decarboxylation of orotidine monophosphate (OMP) to form uridine monophosphate (UMP). The function of this enzyme is essential to the de novo biosynthesis of the pyrimidine nucleotides uridine triphosphate, cytidine triphosphate, and thymidine triphosphate. OMP decarboxylase has been a frequent target for scientific investigation because of its demonstrated extreme catalytic efficiency and its usefulness as a selection marker for yeast strain engineering.
Catalysis
OMP decarboxylase is known for being an extraordinarily efficient catalyst capable of accelerating the uncatalyzed reaction rate by a factor of 1017. To put this in perspective, the uncatalysed reaction which would take 78 million years to convert half the reactants into products is accelerated to 18 milliseconds when catalyzed by OMP decarboxylase.[2] This extreme enzymatic efficiency is especially interesting because OMP decarboxylases uses no cofactor and contains no metal sites[3] or prosthetic groups.[4] The catalysis relies on a handful of charged amino acid residues positioned within the active site of the enzyme.
The exact mechanism by which OMP decarboxylase catalyzes its reaction has been a subject of rigorous scientific investigation. The driving force for the loss of the carboxyl linked to the C6 of the pyrimidine ring comes from the close proximity of an aspartate residue carboxyl group in the enzyme's active site, which destabilizes the ground state relative to the transition state of the uncatalyzed reaction. There have been multiple hypotheses about what form the transition state takes before protonation of the C6 carbon occurs to yield the final product. Many studies investigated the binding of a potent inhibitor of OMP decarboxylase, 6-hydroxy uridine monophosphate (BMP, a barbituric acid derivative), within the active site, to identify which essential amino acid residues are directly involved with stabilization of the transition state. (See figure of enzyme bound to BMP) Several mechanisms for enzymatic decarboxylation of OMP have been proposed, including protonation at O2 to form a zwitterionic species as an intermediate,[6] anion stabilization of O4,[7] or nucleophilic attack at C5.[8] Current consensus suggests that the mechanism proceeds through a stabilized carbanion at the C6 after loss of carbon dioxide. This mechanism was suggested from studies investigating kinetic isotope effects in conjunction with competitive inhibition and active site mutagenesis.[9][10][11][12] In this mechanism the short-lived carbanion species is stabilized by a nearby lysine residue, before it is quenched by a proton. (See schematic of catalytic mechanism) The intermediacy of a highly basic vinyl carbanion not benefiting from electronic stabilization is rare in an enzymatic system and in biological systems in general. Remarkably, the enzyme microenvironment helps stabilize the carbanion considerably. The pKaH of the enzyme-bound carbanionic intermediate was measured to be less than or equal to 22 based on deuterium exchange studies. While still highly basic, the corresponding pKaH of the free carbanionic intermediate is estimated to be much higher, around 30-34 (based on measurements on the analogous 1,3-dimethyluracil), leading to the conclusion that the enzyme stabilizes the carbanion by at least 14 kcal/mol.[12]
Relation to UMP synthase
In yeast and bacteria, OMP decarboxylase is a single-function enzyme. However, in mammals, OMP decarboxylase is part of a single protein with two catalytic activities. This bifunctional enzyme is named UMP synthase and it also catalyzes the preceding reaction in pyrimidine nucleotide biosynthesis, the transfer of ribose 5-phosphate from 5-phosphoribosyl-1-pyrophosphate to orotate to form OMP. In organisms utilizing OMP decarboxylase, this reaction is catalyzed by orotate phosphoribosyltransferase.[14]
Importance in yeast genetics
Mutations in the gene encoding OMP decarboxylase in yeast (URA3) leads to auxotrophy in uracil. In addition, a function OMP decarboxylase renders yeast strains sensitive to the molecule 5-fluoroorotic acid (5-FOA).[15] The establishment of the URA3 gene as a selection marker with both positive and negative selection strategies has made the controlled expression of OMP decarboxylase a significant laboratory tool for the investigation of yeast genetics.
See also
References
- ↑ PDB: 1EIX; "Structural basis for the catalytic mechanism of a proficient enzyme: orotidine 5'-monophosphate decarboxylase". Biochemistry 39 (15): 4217–24. April 2000. doi:10.1021/bi992952r. PMID 10757968.
- ↑ "A proficient enzyme". Science 267 (5194): 90–3. January 1995. doi:10.1126/science.7809611. PMID 7809611.
- ↑ "Activity of yeast orotidine-5'-phosphate decarboxylase in the absence of metals". J. Biol. Chem. 274 (34): 23841–3. August 1999. doi:10.1074/jbc.274.34.23841. PMID 10446147.
- ↑ "Catalytic proficiency: the unusual case of OMP decarboxylase". Annu. Rev. Biochem. 71: 847–85. 2002. doi:10.1146/annurev.biochem.71.110601.135446. PMID 12045113.
- ↑ "Crystal structures of inhibitor complexes reveal an alternate binding mode in orotidine-5'-monophosphate decarboxylase". J. Biol. Chem. 277 (31): 28080–7. August 2002. doi:10.1074/jbc.M202362200. PMID 12011084.
- ↑ "Mechanism of decarboxylation of 1,3-dimethylorotic acid. A model for orotidine 5'-phosphate decarboxylase". J Am Chem Soc 98 (12): 3601–6. 1976. doi:10.1021/ja00428a035. PMID 1270703.
- ↑ "A proficient enzyme revisited: the predicted mechanism for orotidine monophosphate decarboxylase". Science 276 (5314): 942–5. May 1997. doi:10.1126/science.276.5314.942. PMID 9139656.
- ↑ Silverman, R.B.; Groziak, M.P. (1982). "Model Chemistry for a Covalent Mechanism of Action of Orotidine 5'-Phosphate Decarboxylase". J. Am. Chem. Soc. 104 (23): 6434–6439. doi:10.1021/ja00387a047.
- ↑ Lee, Jeehiun K; Tantillo, Dean J (2004-06-25). Orotidine Monophosphate Decarboxylase: A Mechanistic Dialogue. ISBN 9783540205661.
- ↑ "Determination of the Mechanism of Orotidine 5'-Monophosphate Decarboxylase by Isotope Effects". Biochemistry 39 (16): 4569–4574. 2000. doi:10.1021/bi000376p. PMID 10769111.
- ↑ "Product Deuterium Isotope Effect for Orotidine 5'-Monophosphate Decarboxylase: Evidence for the Existence of a Short-Lived Carbanion Intermediate". J. Am. Chem. Soc. 129 (43): 12946–7. October 2007. doi:10.1021/ja076222f. PMID 17918849.
- ↑ 12.0 12.1 "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". J. Am. Chem. Soc. 130 (5): 1574–5. February 2008. doi:10.1021/ja710384t. PMID 18186641.
- ↑ "Carbon isotope effect study on orotidine 5'-monophosphate decarboxylase: support for an anionic intermediate". Biochemistry 47 (2): 798–803. January 2008. doi:10.1021/bi701664n. PMID 18081312.
- ↑ "Intrinsic activity and stability of bifunctional human UMP synthase and its two separate catalytic domains, orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase". J Biol Chem 271 (18): 10704–10708. 1996. doi:10.1074/jbc.271.18.10704. PMID 8631878.
- ↑ "A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance". Mol Gen Genet 197 (2): 345–346. 1984. doi:10.1007/BF00330984. PMID 6394957.
 |  |
