An examination of the relationship between active site loop size and thermodynamic activation parameters for orotidine 5'-monophosphate decarboxylase from mesophilic and thermophilic organisms.
ABSTRACT: Closure of the active site phosphate gripper loop of orotidine 5'-monophosphate decarboxylase from Saccharomyces cerevisiae (ScOMPDC) over the bound substrate orotidine 5'-monophosphate (OMP) activates the bound substrate for decarboxylation by at least 10(4)-fold [Amyes, T. L., Richard, J. P., and Tait, J. J. (2005) J. Am. Chem. Soc. 127, 15708-15709]. The 19-residue phosphate gripper loop of the mesophilic ScOMPDC is much larger than the nine-residue loop at the ortholog from the thermophile Methanothermobacter thermautotrophicus (MtOMPDC). This difference in loop size results in a small decrease in the total intrinsic phosphate binding energy of the phosphodianion group of OMP from 11.9 to 11.6 kcal/mol, along with a modest decrease in the extent of activation by phosphite dianion of decarboxylation of the truncated substrate 1-(beta-D-erythrofuranosyl)orotic acid. The activation parameters DeltaH(double dagger) and DeltaS(double dagger) for k(cat) for decarboxylation of OMP are 3.6 kcal/mol and 10 cal K(-1) mol(-1) more positive, respectively, for MtOMPDC than for ScOMPDC. We suggest that these differences are related to the difference in the size of the active site loops at the mesophilic ScOMPDC and the thermophilic MtOMPDC. The greater enthalpic transition state stabilization available from the more extensive loop-substrate interactions for the ScOMPDC-catalyzed reaction is largely balanced by a larger entropic requirement for immobilization of the larger loop at this enzyme.
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: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 -CH2OH 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 kcat/ Km 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 ? G? 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.
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.
Project description:Kinetic parameters kex (s-1) and kex/Kd (M-1 s-1) are reported for exchange for deuterium in D2O of the C-6 hydrogen of 5-fluororotidine 5'-monophosphate (FUMP) catalyzed by the Q215A, Y217F, and Q215A/Y217F variants of yeast orotidine 5'-monophosphate decarboxylase (ScOMPDC) at pD 8.1, and by the Q215A variant at pD 7.1-9.3. The pD rate profiles for wildtype ScOMPDC and the Q215A variant are identical, except for a 2.5 log unit downward displacement in the profile for the Q215A variant. The Q215A, Y217F and Q215A/Y217F substitutions cause 1.3-2.0 kcal/mol larger increases in the activation barrier for wildtype ScOMPDC-catalyzed deuterium exchange compared with decarboxylation, because of the stronger apparent side chain interaction with the transition state for the deuterium exchange reaction. The stabilization of the transition state for the OMPDC-catalyzed deuterium exchange reaction of FUMP is ca. 19 kcal/mol smaller than the transition state for decarboxylation of OMP, and ca. 8 kcal/mol smaller than for OMPDC-catalyzed deprotonation of FUMP to form the vinyl carbanion intermediate common to OMPDC-catalyzed reactions OMP/FOMP and UMP/FUMP. We propose that ScOMPDC shows similar stabilizing interactions with the common portions of decarboxylation and deprotonation transition states that lead to formation of this vinyl carbanion intermediate, and that there is a large ca. (19-8) = 11 kcal/mol stabilization of the former transition state from interactions with the nascent CO2 of product. The effects of Q215A and Y217F substitutions on kcat/Km for decarboxylation of OMP are expressed mainly as an increase in Km for the reactions catalyzed by the variant enzymes, while the effects on kex/Kd for deuterium exchange are expressed mainly as an increase in kex. This shows that the Q215 and Y217 side chains stabilize the Michaelis complex to OMP for the decarboxylation reaction, compared with the complex to FUMP for the deuterium exchange reaction. These results provide strong support for the conclusion that interactions which stabilize the transition state for ScOMPDC-catalyzed decarboxylation at a nonpolar enzyme active site dominate over interactions that destabilize the ground-state Michaelis complex.
Project description: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 k(cat) 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-?-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 (k(cat))(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 (K(m))(obs).
Project description:Triosephosphate isomerase (TIM), glycerol 3-phosphate dehydrogenase, and orotidine 5'-monophosphate decarboxylase each use the binding energy from the interaction of phosphite dianion with a flexible phosphate gripper loop to activate a second, phosphodianion-truncated, substrate towards enzyme-catalyzed proton transfer, hydride transfer, and decarboxylation, respectively. Studies on TIM suggest that the most important general effect of loop closure over the substrate phosphodianion, and the associated conformational changes, is to extrude water from the enzyme active site. This should cause a decrease in the effective active-site dielectric constant, and an increase in transition state stabilization from enhanced electrostatic interactions with polar amino acid side chains. The most important specific effect of these conformational changes is to increase the basicity of the carboxylate side chain of the active site glutamate base by its placement in a 'hydrophobic cage'.
Project description:The reaction catalyzed by orotidine 5'-monophosphate decarboxylase (OMPDC) involves a stabilized anionic intermediate, although the structural basis for the rate acceleration (k(cat)/k(non), 7.1 x 10(16)) and proficiency [(k(cat)/K(M))/k(non), 4.8 x 10(22) M(-1)] is uncertain. That the OMPDCs from Methanothermobacter thermautotrophicus (MtOMPDC) and Saccharomyces cerevisiae (ScOMPDC) catalyze the exchange of H6 of the UMP product with solvent deuterium allows an estimate of a lower limit on the rate acceleration associated with stabilization of the intermediate and its flanking transition states (>or=10(10)). The origin of the "missing" contribution, <or=10(7) ( approximately 10(17) total - >or=10(10)), is of interest. Based on structures of liganded complexes, unfavorable electrostatic interactions between the substrate carboxylate group and a proximal Asp (Asp 70 in MtOMPDC and Asp 91 in ScOMPDC) have been proposed to contribute to the catalytic efficiency [Wu, N., Mo, Y., Gao, J., and Pai, E. F. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 2017-2022]. We investigated that hypothesis by structural and functional characterization of the D70N and D70G mutants of MtOMPDC and the D91N mutant of ScOMPDC. The substitutions for Asp 70 in MtOMPDC significantly decrease the value of k(cat) for decarboxylation of FOMP (a more reactive substrate analogue) but have little effect on the value of k(ex) for exchange of H6 of FUMP with solvent deuterium; the structures of wild-type MtOMPDC and its mutants are superimposable when complexed with 6-azaUMP. In contrast, the D91N mutant of ScOMPDC does not catalyze exchange of H6 of FUMP; the structures of wild-type ScOMPDC and its D91N mutant are not superimposable when complexed with 6-azaUMP, with differences in both the conformation of the active site loop and the orientation of the ligand vis a vis the active site residues. We propose that the differential effects of substitutions for Asp 70 of MtOMPDC on decarboxylation and exchange provide additional evidence for a carbanionic intermediate as well as the involvement of Asp 70 in substrate destabilization.
Project description:Orotidine 5'-monophosphate decarboxylase (OMPDC) catalyzes the decarboxylation of 5-fluoroorotate (FO) with kcat/Km = 1.4 × 10-7 M-1 s-1. Combining this and related kinetic parameters shows that the 31 kcal/mol stabilization of the transition state for decarboxylation of OMP provided by OMPDC represents the sum of 11.8 and 10.6 kcal/mol stabilization by the substrate phosphodianion and the ribosyl ring, respectively, and an 8.6 kcal/mol stabilization from the orotate ring. The transition state for OMPDC-catalyzed decarboxylation of FO is stabilized by 5.2, 7.2, and 9.0 kcal/mol, respectively, by 1.0 M phosphite dianion, d-glycerol 3-phosphate and d-erythritol 4-phosphate. The stabilization is due to the utilization of binding interactions of the substrate fragments to drive an enzyme conformational change, which locks the orotate ring of the whole substrate, or the substrate pieces in a caged complex. We propose that enzyme-activation is a possible, and perhaps probable, consequence of any substrate-induced enzyme conformational change.
Project description:Kinetic analysis of decarboxylation catalyzed by S154A, Q215A, and S154A/Q215A mutant yeast orotidine 5'-monophosphate decarboxylases with orotidine 5'-monophosphate (OMP) and with a truncated nucleoside substrate (EO) activated by phosphite dianion shows (1) the side chain of Ser-154 stabilizes the transition state through interactions with the pyrimidine rings of OMP or EO, (2) the side chain of Gln-215 interacts with the phosphodianion group of OMP or with phosphite dianion, and (3) the interloop hydrogen bond between the side chains of Ser-154 and Gln-215 orients the amide side chain of Gln-215 to interact with the phosphodianion group of OMP or with phosphite dianion.
Project description:The kinetic parameters for activation of yeast triosephosphate isomerase (ScTIM), yeast orotidine monophosphate decarboxylase (ScOMPDC), and human liver glycerol 3-phosphate dehydrogenase (hlGPDH) for catalysis of reactions of their respective phosphodianion truncated substrates are reported for the following oxydianions: HPO3(2-), FPO3(2-), S2O3(2-), SO4(2-) and HOPO3(2-). Oxydianions bind weakly to these unliganded enzymes and tightly to the transition state complex (E·S(‡)), with intrinsic oxydianion Gibbs binding free energies that range from -8.4 kcal/mol for activation of hlGPDH-catalyzed reduction of glycolaldehyde by FPO3(2-) to -3.0 kcal/mol for activation of ScOMPDC-catalyzed decarboxylation of 1-?-d-erythrofuranosyl)orotic acid by HOPO3(2-). Small differences in the specificity of the different oxydianion binding domains are observed. We propose that the large -8.4 kcal/mol and small -3.8 kcal/mol intrinsic oxydianion binding energy for activation of hlGPDH by FPO3(2-) and S2O3(2-), respectively, compared with activation of ScTIM and ScOMPDC reflect stabilizing and destabilizing interactions between the oxydianion -F and -S with the cationic side chain of R269 for hlGPDH. These results are consistent with a cryptic function for the similarly structured oxydianion binding domains of ScTIM, ScOMPDC and hlGPDH. Each enzyme utilizes the interactions with tetrahedral inorganic oxydianions to drive a conformational change that locks the substrate in a caged Michaelis complex that provides optimal stabilization of the different enzymatic transition states. The observation of dianion activation by stabilization of active caged Michaelis complexes may be generalized to the many other enzymes that utilize substrate binding energy to drive changes in enzyme conformation, which induce tight substrate fits.