title: Catalysis by orotidine 5'-monophosphate decarboxylase: effect of 5-fluoro and 4'-substituents on the decarboxylation of two-part substrates.
abstract: The syntheses of two novel truncated analogs of the natural substrate orotidine 5'-monophosphate (OMP) for orotidine 5'-monophosphate decarboxylase (OMPDC) with enhanced reactivity toward decarboxylation are reported: 1-(β-d-erythrofuranosyl)-5-fluoroorotic acid (FEO) and 5'-deoxy-5-fluoroorotidine (5'-dFO). A comparison of the second-order rate constants for the OMPDC-catalyzed decarboxylations of FEO (10 M⁻¹ s⁻¹) and 1-(β-d-erythrofuranosyl)orotic acid (EO, 0.026 M⁻¹ s⁻¹) shows that the vinyl carbanion-like transition state is stabilized by 3.5 kcal/mol by interactions with the 5-F substituent of FEO. The OMPDC-catalyzed decarboxylations of FEO and EO are both activated by exogenous phosphite dianion (HPO₃²⁻), but the 5-F substituent results in only a 0.8 kcal stabilization of the transition state for the phosphite-activated reaction of FEO. This provides strong evidence that the phosphite-activated OMPDC-catalyzed reaction of FEO is not limited by the chemical step of decarboxylation of the enzyme-bound substrate. Evidence is presented that there is a change in the rate-limiting step from the chemical step of decarboxylation for the phosphite-activated reaction of EO, to closure of the phosphate gripper loop and an enzyme conformational change at the ternary E•FEO•HPO₃²⁻ complex for the reaction of FEO. The 4'-CH₃ and 4'-CH₂OH groups of 5'-dFO and orotidine, respectively, result in identical destabilizations of the transition state for the unactivated decarboxylation of 2.9 kcal/mol. By contrast, the 4'-CH₃ group of 5'-dFO and the 4'-CH₂OH group of orotidine result in very different 4.7 and 8.3 kcal/mol destabilizations of the transition state for the phosphite-activated decarboxylation. Here, the destabilizing effect of the 4'-CH₃ substituent at 5'-dFO is masked by the rate-limiting conformational change that depresses the third-order rate constant for the phosphite-activated reaction of the parent substrate FEO.
authors.affiliation: Department of Chemistry, University at Buffalo, SUNY, Buffalo, NY 14260-3000, USA.
authors.forename: Bogdana
authors.initials: B
authors.lastname: Goryanova
authors.forename: Krisztina
authors.initials: K
authors.lastname: Spong
authors.forename: Tina L
authors.initials: TL
authors.lastname: Amyes
authors.forename: John P
authors.initials: JP
authors.lastname: Richard
mesh_terms: D055162:Biocatalysis; D002021:Buffers; D003653:Decarboxylation; D004789:Enzyme Activation; D004795:Enzyme Stability; D007700:Kinetics; D050505:Mutant Proteins / Q000378:metabolism; D009963:Orotic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000235:genetics / Q000378:metabolism*; D009994:Osmolar Concentration; D017905:Phosphites / Q000737:chemistry; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014529:Uridine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D014542:Uridine Monophosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism
chemical_list: C578856:1-(beta-D-erythrofuranosyl)-5-fluoroorotic acid; C578857:1-(beta-D-erythrofuranosyl)orotic acid; C578855:5'-deoxy-5-fluoroorotidine; D002021:Buffers; D050505:Mutant Proteins; D017905:Phosphites; D029701:Saccharomyces cerevisiae Proteins; C005417:orotidylic acid; D009963:Orotic Acid; C008714:orotidine; D014542:Uridine Monophosphate; D009964:Orotidine-5'-Phosphate Decarboxylase; D014529:Uridine