Role of a guanidinium cation-phosphodianion pair in stabilizing the vinyl carbanion intermediate of orotidine 5'-phosphate decarboxylase-catalyzed reactions.
The side chain cation of Arg235 provides a 5.6 and 2.6 kcal/mol stabilization of the transition states for orotidine 5'-monophosphate (OMP) decarboxylase (OMPDC) from Saccharomyces cerevisiae catalyzed reactions of OMP and 5-fluoroorotidine 5'-monophosphate (FOMP), respectively, a 7.2 kcal/mol stabilization of the vinyl carbanion-like transition state for enzyme-catalyzed exchange of the C-6 proton of 5-fluorouridine 5'-monophosphate (FUMP), but no stabilization of the transition states for enzyme-catalyzed decarboxylation of truncated substrates 1-(β-d-erythrofuranosyl)orotic acid and 1-(β-d-erythrofuranosyl) 5-fluorouracil. These observations show that the transition state stabilization results from formation of a protein cation-phosphodianion pair, and that there is no detectable stabilization from an interaction between the side chain and the pyrimidine ring of substrate. The 5.6 kcal/mol side chain interaction with the transition state for the decarboxylation reaction is 50% of the total 11.2 kcal/mol transition state stabilization by interactions with the phosphodianion of OMP, whereas the 7.2 kcal/mol side chain interaction with the transition state for the deuterium exchange reaction is a larger 78% of the total 9.2 kcal/mol transition state stabilization by interactions with the phosphodianion of FUMP. The effect of the R235A mutation on the enzyme-catalyzed deuterium exchange is expressed predominantly as a change in the turnover number kex, whereas the effect on the enzyme-catalyzed decarboxylation of OMP is expressed predominantly as a change in the Michaelis constant Km. These results are rationalized by a mechanism in which the binding of OMP, compared with that for FUMP, provides a larger driving force for conversion of OMPDC from an inactive open conformation to a productive, active, closed conformation.
Department of Chemistry, University at Buffalo , Buffalo, New York 14260, United States.
Bogdana
B
Goryanova
Lawrence M
LM
Goldman
Tina L
TL
Amyes
John A
JA
Gerlt
John P
JP
Richard
D018360:Crystallography, X-Ray; D003653:Decarboxylation; D003903:Deuterium; D019791:Guanidine / Q000737:chemistry*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D009154:Mutation / Q000235:genetics; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D012265:Ribonucleotides / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013379:Substrate Specificity; D014542:Uridine Monophosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D014544:Uridine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
C019391:5-fluoroorotidine 5'-phosphate; D012265:Ribonucleotides; C005417:orotidylic acid; C025635:5-fluorouridine 5'-triphosphate; D003903:Deuterium; D014542:Uridine Monophosphate; D009964:Orotidine-5'-Phosphate Decarboxylase; D019791:Guanidine; D014544:Uridine Triphosphate