Enzyme architecture

Deconstruction of the enzyme-activating phosphodianion interactions of orotidine 5′-monophosphate decarboxylase

Lawrence M. Goldman, Tina L. Amyes, Bogdana Goryanova, John Alan Gerlt, John P. Richard

Research output: Contribution to journalArticle

Abstract

The mechanism for activation of orotidine 5′-monophosphate decarboxylase (OMPDC) by interactions of side chains from Gln215 and Try217 at a gripper loop and R235, adjacent to this loop, with the phosphodianion of OMP was probed by determining the kinetic parameters kcat and K m for all combinations of single, double, and triple Q215A, Y217F, and R235A mutations. The 12 kcal/mol intrinsic binding energy of the phosphodianion is shown to be equal to the sum of the binding energies of the side chains of R235 (6 kcal/mol), Q215 (2 kcal/mol), Y217 (2 kcal/mol), and hydrogen bonds to the G234 and R235 backbone amides (2 kcal/mol). Analysis of a triple mutant cube shows small (ca. 1 kcal/mol) interactions between phosphodianion gripper side chains, which are consistent with steric crowding of the side chains around the phosphodianion at wild-type OMPDC. These mutations result in the same change in the activation barrier to the OMPDC-catalyzed reactions of the whole substrate OMP and the substrate pieces (1-β-d-erythrofuranosyl)orotic acid (EO) and phosphite dianion. This shows that the transition states for these reactions are stabilized by similar interactions with the protein catalyst. The 12 kcal/mol intrinsic phosphodianion binding energy of OMP is divided between the 8 kcal/mol of binding energy, which is utilized to drive a thermodynamically unfavorable conformational change of the free enzyme, resulting in an increase in (kcat)obs for OMPDC-catalyzed decarboxylation of OMP, and the 4 kcal/mol of binding energy, which is utilized to stabilize the Michaelis complex, resulting in a decrease in (Km)obs.

Original languageEnglish (US)
Pages (from-to)10156-10165
Number of pages10
JournalJournal of the American Chemical Society
Volume136
Issue number28
DOIs
StatePublished - Jul 16 2014

Fingerprint

Carboxy-Lyases
Binding energy
Enzymes
enzyme
Grippers
energy
Orotic Acid
Phosphites
mutation
Mutation
Decarboxylation
Chemical activation
substrate
decarboxylation
Amides
Hydrogen
Substrates
Kinetic parameters
Hydrogen bonds
catalyst

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Enzyme architecture : Deconstruction of the enzyme-activating phosphodianion interactions of orotidine 5′-monophosphate decarboxylase. / Goldman, Lawrence M.; Amyes, Tina L.; Goryanova, Bogdana; Gerlt, John Alan; Richard, John P.

In: Journal of the American Chemical Society, Vol. 136, No. 28, 16.07.2014, p. 10156-10165.

Research output: Contribution to journalArticle

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