Archie C. Reyes

The Activating Oxydianion Binding Domain for Enzyme-Catalyzed Proton Transfer, Hydride Transfer, and Decarboxylation: Specificity and Enzyme Architecture

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: HPO32–, FPO32–, S2O32–, SO42– and HOPO32–. 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 FPO32– to −3.0 kcal/mol for activation of ScOMPDC-catalyzed decarboxylation of 1-β-d-erythrofuranosyl)orotic acid by HOPO32–. 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 FPO32– and S2O32–, 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.

Enzyme Architecture: Optimization of Transition State Stabilization from a Cation–Phosphodianion Pair

The side chain cation of R269 lies at the surface of l-glycerol 3-phosphate dehydrogenase (GPDH) and forms an ion pair to the phosphodianion of substrate dihydroxyacetone phosphate (DHAP), which is buried at the nonpolar protein interior. The R269A mutation of GPDH results in a 110-fold increase in Km (2.8 kcal/mol effect) and a 41 000-fold decrease in kcat (6.3 kcal/mol effect), which corresponds to a 9.1 kcal/mol destabilization of the transition state for GPDH-catalyzed reduction of DHAP by NADH. There is a 6.7 kcal/mol stabilization of the transition state for the R269A mutant GPDH-catalyzed reaction by 1.0 M guanidinium ion, and the transition state for the reaction of the substrate pieces is stabilized by an additional 2.4 kcal/mol by their covalent attachment at wildtype GPDH. These results provide strong support for the proposal that GPDH invests the 11 kcal/mol intrinsic phosphodianion binding energy of DHAP in trapping the substrate at a nonpolar active site, where strong electrostatic interactions are favored, and obtains a 9 kcal/mol return from stabilizing interactions between the side chain cation and transition state trianion. We propose a wide propagation for the catalytic motif examined in this work, which enables strong transition state stabilization from enzyme–phosphodianion pairs.

Enzyme Architecture: Self-Assembly of Enzyme and Substrate Pieces of Glycerol-3-Phosphate Dehydrogenase into a Robust Catalyst of Hydride Transfer

The stabilization of the transition state for hlGPDH-catalyzed reduction of DHAP due to the action of the phosphodianion of DHAP and the cationic side chain of R269 is between 12.4 and 17 kcal/mol. The R269A mutation of glycerol-3-phosphate dehydrogenase (hlGPDH) results in a 9.1 kcal/mol destabilization of the transition state for enzyme-catalyzed reduction of dihydroxyacetone phosphate (DHAP) by NADH, and there is a 6.7 kcal/mol stabilization of this transition state by 1.0 M guanidine cation (Gua+) [J. Am. Chem. Soc.2015, 137, 5312–5315]. The R269A mutant shows no detectable activity toward reduction of glycolaldehyde (GA), or activation of this reaction by 30 mM HPO32–. We report the unprecedented self-assembly of R269A hlGPDH, dianions (X2– = FPO32–, HPO32–, or SO42–), Gua+ and GA into a functioning catalyst of the reduction of GA, and fourth-order reaction rate constants kcat/KGAKXKGua. The linear logarithmic correlation (slope = 1.0) between values of kcat/KGAKX for dianion activation of wildtype hlGPDH-catalyzed reduction of GA and kcat/KGAKXKGua shows that the electrostatic interaction between exogenous dianions and the side chain of R269 is not significantly perturbed by cutting hlGPDH into R269A and Gua+ pieces. The advantage for connection of hlGPDH (R269A mutant + Gua+) and substrate pieces (GA + HPi) pieces, (ΔGS‡)HPi+E+Gua = 5.6 kcal/mol, is nearly equal to the sum of the advantage to connection of the substrate pieces, (ΔGS‡)GA+HPi = 3.3 kcal/mol, for wildtype hlGPDH-catalyzed reaction of GA + HPi, and for connection of the enzyme pieces, (ΔGS‡)E+Gua = 2.4 kcal/mol, for Gua+ activation of the R269A hlGPDH-catalyzed reaction of DHAP.

Structure–Reactivity Effects on Intrinsic Primary Kinetic Isotope Effects for Hydride Transfer Catalyzed by Glycerol-3-phosphate Dehydrogenase

Primary deuterium kinetic isotope effects (1°DKIE) on (kcat/KGA, M–1 s–1) for dianion (X2–) activated hydride transfer from NADL to glycolaldehyde (GA) catalyzed by glycerol-3-phosphate dehydrogenase were determined over a 2100-fold range of enzyme reactivity: (X2–, 1°DKIE); FPO32–, 2.8 ± 0.1; HPO32–, 2.5 ± 0.1; SO42–, 2.8 ± 0.2; HOPO32–, 2.5 ± 0.1; S2O32–, 2.9 ± 0.1; unactivated; 2.4 ± 0.2. Similar 1°DKIEs were determined for kcat. The observed 1°DKIEs are essentially independent of changes in enzyme reactivity with changing dianion activator. The results are consistent with (i) fast and reversible ligand binding; (ii) the conclusion that the observed 1°DKIEs are equal to the intrinsic 1°DKIE on hydride transfer from NADL to GA; (iii) similar intrinsic 1°DKIEs on GPDH-catalyzed reduction of the substrate pieces and the whole physiological substrate dihydroxyacetone phosphate. The ground-state binding interactions for different X2– are similar, but there are large differences in the transition state interactions for different X2–. The changes in transition state binding interactions are expressed as changes in kcat and are proposed to represent changes in stabilization of the active closed form of GPDH. The 1°DKIEs are much smaller than observed for enzyme-catalyzed hydrogen transfer that occurs mainly by quantum-mechanical tunneling.

Enzyme Architecture: A Startling Role for Asn270 in Glycerol 3-Phosphate Dehydrogenase-Catalyzed Hydride Transfer

The side chains of R269 and N270 interact with the phosphodianion of dihydroxyacetone phosphate (DHAP) bound to glycerol 3-phosphate dehydrogenase (GPDH). The R269A, N270A, and R269A/N270A mutations of GPDH result in 9.1, 5.6, and 11.5 kcal/mol destabilization, respectively, of the transition state for GPDH-catalyzed reduction of DHAP by the reduced form of nicotinamide adenine dinucleotide. The N270A mutation results in a 7.7 kcal/mol decrease in the intrinsic phosphodianion binding energy, which is larger than the 5.6 kcal/mol effect of the mutation on the stability of the transition state for reduction of DHAP; a 2.2 kcal/mol stabilization of the transition state for unactivated hydride transfer to the truncated substrate glycolaldehyde (GA); and a change in the effect of phosphite dianion on GPDH-catalyzed reduction of GA, from strongly activating to inhibiting. The N270A mutation breaks the network of hydrogen bonding side chains, Asn270, Thr264, Asn205, Lys204, Asp260, and Lys120, which connect the dianion activation and catalytic sites of GPDH. We propose that this disruption dramatically alters the performance of GPDH at these sites.

Enzyme Architecture: The Role of a Flexible Loop in Activation of Glycerol-3-phosphate Dehydrogenase for Catalysis of Hydride Transfer

The side chain of Q295 of glycerol-3-phosphate dehydrogenase from human liver (hlGPDH) lies in a flexible loop, that folds over the phosphodianion of substrate dihydroxyacetone phosphate (DHAP). Q295 interacts with the side-chain cation from R269, which is ion-paired to the substrate phosphodianion. Kinetic parameters kcat/Km (M–1 s–1) and kcat/KGAKHPi (M–2 s–1) were determined, respectively, for catalysis of the reduction of DHAP and for dianion activation of catalysis of reduction of glycolaldehyde (GA) catalyzed by wild-type, Q295G, Q295S, Q295A, and Q295N mutants of hlGPDH. These mutations result in up to a 150-fold decrease in (kcat/Km)DHAP and up to a 2.7 kcal/mol decrease in the intrinsic phosphodianion binding energy. The data define a linear correlation with slope 1.1, between the intrinsic phosphodianion binding energy and the intrinsic phosphite dianion binding energy for activation of hlGPDH-catalyzed reduction of GA, that demonstrates a role for Q295 in optimizing this dianion binding energy. The R269A mutation of wild-type GPDH results in a 9.1 kcal/mol destabilization of the transition state for reduction of DHAP, but the same R269A mutation of N270A and Q295A mutants result in smaller 5.9 and 4.9 kcal/mol transition-state destabilization. Similarly, the N270A or Q295A mutations of R269A GPDH each result in large falloffs in the efficiency of rescue of the R269A mutant by guanidine cation. We conclude that N270, which interacts for the substrate phosphodianion and Q295, which interacts with the guanidine side chain of R269, function to optimize the apparent transition-state stabilization provided by the cationic side chain of R269.

Orotidine 5′-Monophosphate Decarboxylase: Probing the Limits of the Possible for Enzyme Catalysis

Conspectus 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 pKa that is associated with ligand binding. The binding of UMP to OMPDC results in an unusually large >12 unit decrease in the pKa = 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.

Primary Deuterium Kinetic Isotope Effects: A Probe for the Origin of the Rate Acceleration for Hydride Transfer Catalyzed by Glycerol-3-Phosphate Dehydrogenase

Large primary deuterium kinetic isotope effects (1° DKIEs) on enzyme-catalyzed hydride transfer may be observed when the transferred hydride tunnels through the energy barrier. The following 1° DKIEs on kcat/Km and relative reaction driving force are reported for wild-type and mutant glycerol-3-phosphate dehydrogenase (GPDH)-catalyzed reactions of NADL (L = H, D): wild-type GPDH, ΔΔG⧧ = 0 kcal/mol, 1° DKIE = 1.5; N270A, 5.6 kcal/mol, 3.1; R269A, 9.1 kcal/mol, 2.8; R269A + 1.0 M guanidine, 2.4 kcal/mol, 2.7; R269A/N270A, 11.5 kcal/mol, 2.4. Similar 1° DKIEs were observed on kcat. The narrow range of 1° DKIEs (2.4–3.1) observed for a 9.1 kcal/mol change in reaction driving force provides strong evidence that these are intrinsic 1° DKIEs on rate-determining hydride transfer. Evidence is presented that the intrinsic DKIE on wild-type GPDH-catalyzed reduction of DHAP lies in this range. A similar range of 1° DKIEs (2.4–2.9) on (kcat/KGA, M–1 s–1) was reported for dianion-activated hydride transfer from NADL to glycolaldehyde (GA) [Reyes, A. C.; Amyes, T. L.; Richard, J. P. J. Am. Chem. Soc.2016, 138, 14526–14529]. These 1° DKIEs are much smaller than those observed for enzyme-catalyzed hydrogen transfer that occurs mainly by quantum mechanical tunneling. These results support the conclusion that the rate acceleration for GPDH-catalyzed reactions is due to the stabilization of the transition state for hydride transfer by interactions with the protein catalyst. The small 1° DKIEs reported for mutant GPDH-catalyzed and for wild-type dianion-activated reactions are inconsistent with a model where the dianion binding energy is utilized in the stabilization of a tunneling ready state.

Enzyme Architecture: Erection of Active Orotidine 5′-Monophosphate Decarboxylase by Substrate-Induced Conformational Changes

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.