Enzyme Architecture

Breaking Down the Catalytic Cage that Activates Orotidine 5′-Monophosphate Decarboxylase for Catalysis

Archie C. Reyes, David C. Plache, Astrid P. Koudelka, Tina L. Amyes, John Alan Gerlt, John P. Richard

Research output: Contribution to journalArticle

Abstract

We report the results of a study of the catalytic role of a network of four interacting amino acid side chains at yeast orotidine 5′-monophosphate decarboxylase (ScOMPDC), by the stepwise replacement of all four side chains. The H-bond, which links the -CH 2 OH side chain of S154 from the pyrimidine umbrella loop of ScOMPDC to the amide side chain of Q215 in the phosphodianion gripper loop, creates a protein cage for the substrate OMP. The role of this interaction in optimizing transition state stabilization from the dianion gripper side chains Q215, Y217, and R235 was probed by determining the kinetic parameter k cat /K m for 16 enzyme variants, which include all combinations of single, double, triple, and quadruple S154A, Q215A, Y217F, and R235A mutations. The effects of consecutive Q215A, Y217F, and R235A mutations on ΔG for wild-type enzyme-catalyzed decarboxylation sum to 11.6 kcal/mol, but to only 7.6 kcal/mol when starting from S154A mutant. This shows that the S154A mutation results in a (11.6-7.6) = 4.0 kcal/mol decrease in transition state stabilization from interactions with Q215, Y217, and R235. Mutant cycles show that ca. 2 kcal/mol of this 4 kcal/mol effect is from the direct interaction between the S154 and Q215 side chains and that ca. 2 kcal/mol is from a tightening in the stabilizing interactions of the Y217 and R235 side chains. The sum of the effects of individual A154S, A215Q, F217Y and A235R substitutions at the quadruple mutant of ScOMPDC to give the corresponding triple mutants, 5.6 kcal/mol, is much smaller than 16.0 kcal/mol, the sum of the effects of the related four substitutions in wild-type ScOMPDC to give the respective single mutants. The small effect of substitutions at the quadruple mutant is consistent with a large entropic cost to holding the flexible loops of ScOMPDC in the active closed conformation.

Original languageEnglish (US)
Pages (from-to)17580-17590
Number of pages11
JournalJournal of the American Chemical Society
Volume140
Issue number50
DOIs
StatePublished - Dec 19 2018

Fingerprint

Carboxy-Lyases
catalysis
Catalysis
Yeast
Enzymes
Yeasts
yeast
enzyme
Grippers
Substitution reactions
mutation
substitution
Mutation
Stabilization
stabilization
Decarboxylation
decarboxylation
Amides
Kinetic parameters
Conformations

ASJC Scopus subject areas

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

Cite this

Enzyme Architecture : Breaking Down the Catalytic Cage that Activates Orotidine 5′-Monophosphate Decarboxylase for Catalysis. / Reyes, Archie C.; Plache, David C.; Koudelka, Astrid P.; Amyes, Tina L.; Gerlt, John Alan; Richard, John P.

In: Journal of the American Chemical Society, Vol. 140, No. 50, 19.12.2018, p. 17580-17590.

Research output: Contribution to journalArticle

Reyes, Archie C. ; Plache, David C. ; Koudelka, Astrid P. ; Amyes, Tina L. ; Gerlt, John Alan ; Richard, John P. / Enzyme Architecture : Breaking Down the Catalytic Cage that Activates Orotidine 5′-Monophosphate Decarboxylase for Catalysis. In: Journal of the American Chemical Society. 2018 ; Vol. 140, No. 50. pp. 17580-17590.
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