Carbon Isotope Effect Study on Orotidine 5‘-Monophosphate Decarboxylase:  Support for an Anionic Intermediate

Abstract

Orotidine 5'-monophosphate decarboxylase has been heavily examined in recent years due to its enzymatic proficiency, which provides a catalytic enhancement to a reaction rate approximately 1017 times greater than that of the nonenzymatic reaction. Several mechanisms proposed to explain this catalytic enhancement have included covalent addition, ylide or carbene formation, and most recently concerted protonation. All of these mechanisms have circumvented the formation of a high-energy vinyl anionic intermediate. To investigate the presence of an anionic intermediate, 13C isotope effect studies have been performed using the alternate substrate 5-fluoro-OMP (OMP = orotidine 5'-monophosphate). Isotope effects obtained for the wild-type enzyme with OMP and 5-fluoro-OMP are 1.0255 and 1.0106, respectively, corresponding to a decrease of approximately 1.5% for 5-fluoro-OMP. With the K59A enzyme, the intrinisic isotope effects show a similar decrease of approximately 1.9% from 1.0543 with OMP to 1.0356 with 5-fluoro-OMP. This decrease results from the inductive effect of the fluorine, which stabilizes the carbanion intermediate by electron withdrawal and produces a reaction with an earlier transition state. The isotope effect for the decarboxylation of the slow substrate 2'-deoxy-OMP produced a intrinsic isotope effect of nearly 1.0461.

Investigating the role of a backbone to substrate hydrogen bond in OMP decarboxylase using a site-specific amide to ester substitution

“…The rate enhancement is a composite of substrate destabilization and intermediate stabilization (Fig. 1A) (4)(5)(6)(7)(8)(9)(10)(11). The pK a of the UMP product is reduced from 30 to 34 in solution to ≤22 in the active site, requiring significant stabilization of the vinyl anion intermediate (∼14 kcal/mol) (12)(13)(14).…”

Determination of the amino acid sequence requirements for catalysis by the highly proficient orotidine monophosphate decarboxylase

“…Both aspartic acid residues at position 71 and 76 are involved in a highly charged network of interactions providing steric clashes and electrostatic repulsion toward the substrate's carboxyl group leading to substrate destabilization. 15,16,29 Therefore, to assess the contribution of these residue substitutions to the integrity of the charged network, the apparent pK a of each cysteine mutant was determined by using iodoacetamide as a probe to measure deprotonation of the thiol group (Materials and Methods). Both D71C and D76C ODCase are inactivated by treatment with iodoacetamide, whereas the wild-type enzyme is not.…”

“…15,44 Reactions were performed at 25 C in MOPS buffer, pH ¼ 7.2 in a total volume of 0.3 mL and monitored in a Beckman DU 800 spectrophotometer. The wild-type ODCase concentration used was 3.9 nM.…”

“…12 There are several reports that strongly support the existence of a substrate C6 carbanion intermediate during catalysis. [13][14][15] Recent results indicate that Asp70 in the Methanothermobacter thermautotrophicus (Mt) and the analogous Asp91 in the yeast enzyme (E. coli Asp71) provide electronic repulsion and steric hindrance with the carboxylate group to destabilize substrate. 16 This positioning then enables Lys 72 (Mt) and Lys93 (yeast) (E. coli Lys73) to interact preferentially with the negative charge forming at C6 in the transition state.…”

“…The anionic transition state is proposed to be stabilized by the positive charge and then protonated by this active site lysine. [13][14][15] Crystallographic structures of ODCase enzymes with and without bound inhibitors also demonstrate that ligand binding results in substantial motion of the enzyme to close the active site and allow numerous contacts with the ligand. 17,18 The large conformation change involved moves the enzyme from an ''open'' to ''closed'' form.…”