Document Detail


Catalysis by orotidine 5'-monophosphate decarboxylase: effect of 5-fluoro and 4'-substituents on the decarboxylation of two-part substrates.
MedLine Citation:
PMID:  23276261     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Bogdana Goryanova; Krisztina Spong; Tina L Amyes; John P Richard
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2013-01-11
Journal Detail:
Title:  Biochemistry     Volume:  52     ISSN:  1520-4995     ISO Abbreviation:  Biochemistry     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-01-22     Completed Date:  2013-03-14     Revised Date:  2014-01-24    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  537-46     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Biocatalysis
Buffers
Decarboxylation
Enzyme Activation
Enzyme Stability
Kinetics
Mutant Proteins / metabolism
Orotic Acid / analogs & derivatives*,  chemical synthesis,  chemistry,  metabolism
Orotidine-5'-Phosphate Decarboxylase / genetics,  metabolism*
Osmolar Concentration
Phosphites / chemistry
Protein Conformation
Saccharomyces cerevisiae / enzymology*
Saccharomyces cerevisiae Proteins / genetics,  metabolism*
Substrate Specificity
Uridine / analogs & derivatives,  chemistry,  metabolism
Uridine Monophosphate / analogs & derivatives,  chemistry,  metabolism
Grant Support
ID/Acronym/Agency:
GM39754/GM/NIGMS NIH HHS; R01 GM039754/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/1-(beta-D-erythrofuranosyl)-5-fluoroorotic acid; 0/1-(beta-D-erythrofuranosyl)orotic acid; 0/5'-deoxy-5-fluoroorotidine; 0/Buffers; 0/Mutant Proteins; 0/Phosphites; 0/Saccharomyces cerevisiae Proteins; 2149-82-8/orotidylic acid; 61H4T033E5/Orotic Acid; B350MC02GZ/orotidine; E2OU15WN0N/Uridine Monophosphate; EC 4.1.1.23/Orotidine-5'-Phosphate Decarboxylase; WHI7HQ7H85/Uridine
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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