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.
A product deuterium isotope effect (PIE) of 1.0 was determined as the ratio of the yields of [6-(1)H]-uridine 5'-monophosphate (50%) and [6-(2)H]-uridine 5'-monophosphate (50%) from the decarboxylation of orotidine 5'-monophosphate (OMP) in 50/50 (v/v) HOH/DOD catalyzed by orotidine 5'-monophosphate decarboxylase (OMPDC) from Saccharomyces cerevisiae, Methanothermobacter thermautotrophicus, and Escherichia coli. This unitary PIE eliminates a proposed mechanism for enzyme-catalyzed decarboxylation in which proton transfer from Lys-93 to C-6 of OMP provides electrophilic push to the loss of CO(2) in a concerted reaction. We propose that the complete lack of selectivity for the reaction of solvent H and D, which is implied by the value of PIE = 1.0, is enforced by restricted C-N bond rotation of the -CH(2)-NL(3)(+) group of the side chain of Lys-93. A smaller PIE of 0.93 was determined as the ratio of the product yields for OMPDC-catalyzed decarboxylation of 5-fluoroorotidine 5'-monophosphate (5-FOMP) in 50/50 (v/v) HOH/DOD. Mutations on the following important active-site residues of OMPDC from S. cerevisiae have no effect on the PIE on OMPDC-catalyzed decarboxylation of OMP or decarboxylation of 5-FOMP: R235A, Y217A, Q215A, S124A, and S154A/Q215A.
Toth
Krisztina
K
Department of Chemistry, University at Buffalo, Buffalo, New York 14260, USA.
Amyes
Tina L
TL
Wood
Bryant M
BM
Chan
Kui
K
Gerlt
John A
JA
Richard
John P
JP
D055162:Biocatalysis; D003903:Deuterium; D004926:Escherichia coli / Q000201:enzymology; D017013:Methanobacteriaceae / Q000201:enzymology; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012265:Ribonucleotides / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D014542:Uridine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism
C019391:5-fluoroorotidine 5'-phosphate; D012265:Ribonucleotides; C005417:orotidylic acid; D003903:Deuterium; D014542:Uridine Monophosphate; D009964:Orotidine-5'-Phosphate Decarboxylase