Dataset Information

Conformational changes in orotidine 5'-monophosphate decarboxylase: a structure-based explanation for how the 5'-phosphate group activates the enzyme.

ABSTRACT: The binding of a ligand to orotidine 5'-monophosphate decarboxylase (OMPDC) is accompanied by a conformational change from an open, inactive conformation (E(o)) to a closed, active conformation (E(c)). As the substrate traverses the reaction coordinate to form the stabilized vinyl carbanion/carbene intermediate, interactions that destabilize the carboxylate group of the substrate and stabilize the intermediate (in the E(c)·S(‡) complex) are enforced. Focusing on the OMPDC from Methanothermobacter thermautotrophicus, we find the "remote" 5'-phosphate group of the substrate activates the enzyme 2.4 × 10(8)-fold; the activation is equivalently described by an intrinsic binding energy (IBE) of 11.4 kcal/mol. We studied residues in the activation that (1) directly contact the 5'-phosphate group, (2) participate in a hydrophobic cluster near the base of the active site loop that sequesters the bound substrate from the solvent, and (3) form hydrogen bonding interactions across the interface between the "mobile" and "fixed" half-barrel domains of the (?/?)(8)-barrel structure. Our data support a model in which the IBE provided by the 5'-phosphate group is used to allow interactions both near the N-terminus of the active site loop and across the domain interface that stabilize both the E(c)·S and E(c)·S(‡) complexes relative to the E(o)·S complex. The conclusion that the IBE of the 5'-phosphate group provides stabilization to both the E(c)·S and E(c)·S(‡) complexes, not just the E(c)·S(‡) complex, is central to understanding the structural origins of enzymatic catalysis as well as the requirements for the de novo design of enzymes that catalyze novel reactions.

SUBMITTER: Desai BJ

PROVIDER: S-EPMC3549026 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Conformational changes in orotidine 5'-monophosphate decarboxylase: a structure-based explanation for how the 5'-phosphate group activates the enzyme.

Desai Bijoy J BJ Wood B McKay BM Fedorov Alexander A AA Fedorov Elena V EV Goryanova Bogdana B Amyes Tina L TL Richard John P JP Almo Steven C SC Gerlt John A JA

Biochemistry 20121017 43

The binding of a ligand to orotidine 5'-monophosphate decarboxylase (OMPDC) is accompanied by a conformational change from an open, inactive conformation (E(o)) to a closed, active conformation (E(c)). As the substrate traverses the reaction coordinate to form the stabilized vinyl carbanion/carbene intermediate, interactions that destabilize the carboxylate group of the substrate and stabilize the intermediate (in the E(c)·S(‡) complex) are enforced. Focusing on the OMPDC from Methanothermobacte ...[more]

PMID: 23030629

Similar Datasets

Project description:The caged complex between orotidine 5'-monophosphate decarboxylase (ScOMPDC) and 5-fluoroorotidine 5'-monophosphate (FOMP) undergoes decarboxylation ?300 times faster than the caged complex between ScOMPDC and the physiological substrate, orotidine 5'-monophosphate (OMP). Consequently, the enzyme conformational changes required to lock FOMP at a protein cage and release product 5-fluorouridine 5'-monophosphate (FUMP) are kinetically significant steps. The caged form of ScOMPDC is stabilized by interactions between the side chains from Gln215, Tyr217, and Arg235 and the substrate phosphodianion. The control of these interactions over the barrier to the binding of FOMP and the release of FUMP was probed by determining the effect of all combinations of single, double, and triple Q215A, Y217F, and R235A mutations on kcat/Km and kcat for turnover of FOMP by wild-type ScOMPDC; its values are limited by the rates of substrate binding and product release, respectively. The Q215A and Y217F mutations each result in an increase in kcat and a decrease in kcat/Km, due to a weakening of the protein-phosphodianion interactions that favor fast product release and slow substrate binding. The Q215A/R235A mutation causes a large decrease in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of OMP, which are limited by the rate of the decarboxylation step, but much smaller decreases in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of FOMP, which are limited by the rate of enzyme conformational changes. By contrast, the Y217A mutation results in large decreases in kcat/Km for ScOMPDC-catalyzed decarboxylation of both OMP and FOMP, because of the comparable effects of this mutation on rate-determining decarboxylation of enzyme-bound OMP and on the rate-determining enzyme conformational change for decarboxylation of FOMP. We propose that kcat = 8.2 s(-1) for decarboxylation of FOMP by the Y217A mutant is equal to the rate constant for cage formation from the complex between FOMP and the open enzyme, that the tyrosyl phenol group stabilizes the closed form of ScOMPDC by hydrogen bonding to the substrate phosphodianion, and that the phenyl group of Y217 and F217 facilitates formation of the transition state for the rate-limiting conformational change. An analysis of kinetic data for mutant enzyme-catalyzed decarboxylation of OMP and FOMP provides estimates for the rate and equilibrium constants for the conformational change that traps FOMP at the enzyme active site.

Project description:The mystery associated with catalysis by what were once regarded as protein black boxes, diminished with the X-ray crystallographic determination of the three-dimensional structures of enzyme-substrate complexes. The report that several high-resolution X-ray crystal structures of orotidine 5'-monophosphate decarboxylase (OMPDC) failed to provide a consensus mechanism for enzyme-catalyzed decarboxylation of OMP to form uridine 5'-monophosphate, therefore, provoked a flurry of controversy. This controversy was fueled by the enormous 1023-fold rate acceleration for this enzyme, which had " jolted many biochemists' assumptions about the catalytic potential of enzymes." Our studies on the mechanism of action of OMPDC provide strong evidence that catalysis by this enzyme is not fundamentally different from less proficient catalysts, while highlighting important architectural elements that enable a peak level of performance. Many enzymes undergo substrate-induced protein conformational changes that trap their substrates in solvent occluded protein cages, but the conformational change induced by ligand binding to OMPDC is incredibly complex, as required to enable the development of 22 kcal/mol of stabilizing binding interactions with the phosphodianion and ribosyl substrate fragments of OMP. The binding energy from these fragments is utilized to activate OMPDC for catalysis of decarboxylation at the orotate fragment of OMP, through the creation of a tight, catalytically active, protein cage from the floppy, open, unliganded form of OMPDC. Such utilization of binding energy for ligand-driven conformational changes provides a general mechanism to obtain specificity in transition state binding. The rate enhancement that results from the binding of carbon acid substrates to enzymes is partly due to a reduction in the carbon acid p Ka that is associated with ligand binding. The binding of UMP to OMPDC results in an unusually large >12 unit decrease in the p Ka = 29 for abstraction of the C-6 substrate hydrogen, due to stabilization of an enzyme-bound vinyl carbanion, which is also an intermediate of OMPDC-catalyzed decarboxylation. The protein-ligand interactions operate to stabilize the vinyl carbanion at the enzyme active site compared to aqueous solution, rather than to stabilize the transition state for the concerted electrophilic displacement of CO2 by H+ that avoids formation of this reaction intermediate. There is evidence that OMPDC induces strain into the bound substrate. The interaction between the amide side chain of Gln-215 from the phosphodianion gripper loop and the hydroxymethylene side chain of Ser-154 from the pyrimidine umbrella of ScOMPDC position the amide side chain to interact with the phosphodianion of OMP. There are no direct stabilizing interactions between dianion gripper protein side chains Gln-215, Tyr-217, and Arg-235 and the pyrimidine ring at the decarboxylation transition state. Rather these side chains function solely to hold OMPDC in the catalytically active closed conformation. The hydrophobic side chains that line the active site of OMPDC in the region of the departing CO2 product may function to stabilize the decarboxylation transition state by providing hydrophobic solvation of this product.

Dataset Information

Conformational changes in orotidine 5'-monophosphate decarboxylase: a structure-based explanation for how the 5'-phosphate group activates the enzyme.

Publications

Conformational changes in orotidine 5'-monophosphate decarboxylase: a structure-based explanation for how the 5'-phosphate group activates the enzyme.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets