Enzyme Architecture: Remarkably Similar Transition States for Triosephosphate Isomerase-Catalyzed Reactions of the Whole Substrate and the Substrate in Pieces

Zhai, Xiang; Amyes, Tina L.; Richard, John P.

doi:10.1021/ja501103b

Cited by 34 publications

(91 citation statements)

References 41 publications

(92 reference statements)

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“…The model shown in Scheme 3B is strongly supported by the observation that a large number of site-directed mutations of TIM result in the same change in the activation barrier to the reaction of the whole substrate GAP and of the substrate pieces [1- 13 C]-GA + HP i . 60 This result supports the spectator role of HP i , by showing that the transition states for the reactions of the whole substrate and the substrate pieces are stabilized by similar interactions with the protein catalyst, so that by this criteria the transition states have the same structure. 60 …”

Section: Enzyme Activationsupporting

confidence: 64%

“…60 This result supports the spectator role of HP i , by showing that the transition states for the reactions of the whole substrate and the substrate pieces are stabilized by similar interactions with the protein catalyst, so that by this criteria the transition states have the same structure. 60 …”

Section: Enzyme Activationsupporting

confidence: 64%

“…The consequences of this enzyme activation were only recently been revealed in studies on the mechanism for phosphite dianion activation of TIM-catalyzed reactions of glycolaldehyde (GA) and [1- 13 C]-glycolaldehyde ([1- 13 C]-GA) in D 2 O. 23,50,60–64 …”

Section: Enzyme Activationmentioning

confidence: 99%

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Reflections on the catalytic power of a TIM-barrel

Richard¹,

Zhai²,

Malabanan³

2014

Bioorganic Chemistry

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The TIM-barrel fold is described and its propagation throughout the enzyme universe noted. The functions of the individual front loops of the eponymous TIM-barrel of triosephosphate isomerase are presented in a discussion of: (a) Electrophilic catalysis, by amino acid side chains from loops 1 and 4, of abstraction of an α-carbonyl hydrogen from substrate dihydroxyacetone phosphate (DHAP) or D-glyceraldehyde 3-phosphate (DGAP). (b) The engineering of loop 3 to give the monomeric variant monoTIM and the structure and catalytic properties of this monomer. (c) The interaction between loops 6, 7 and 8 and the phosphodianion of DHAP or DGAP. (d) The mechanism by which a ligand-gated conformational change, dominated by motion of loops 6 and 7, activates TIM for catalysis of deprotonation of DHAP or DGAP. (e) The conformational plasticity of TIM, and the utilization of substrate binding energy to “mold” the distorted active site loops of TIM mutants into catalytically active enzymes. The features of the TIM-barrel fold that favor effective protein catalysis are discussed.

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Section: Enzyme Activationsupporting

confidence: 64%

Section: Enzyme Activationsupporting

confidence: 64%

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Reflections on the catalytic power of a TIM-barrel

Richard¹,

Zhai²,

Malabanan³

2014

Bioorganic Chemistry

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“…Although both mutases use a single magnesium ion as the preferred catalytic metal (28,29), they use fundamentally different chemistry: αPGM uses a phosphoserine (30) as an enzyme intermediate, whereas βPGM uses a phosphoaspartate. Classical kinetic studies on rabbit αPGM, based on the binding contributions of various components of the glucose phosphate moiety to the enzyme, were analyzed in terms of a substrateinduced rate effect (31) that has received much support (32)(33)(34)(35). The conservation of the interactions between βPGM and the different inert phosphates in the two TSA complexes is consistent with this analysis.…”

Section: Discussionmentioning

confidence: 64%

α-Fluorophosphonates reveal how a phosphomutase conserves transition state conformation over hexose recognition in its two-step reaction

Jin

Bhattasali

Pellegrini

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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β-Phosphoglucomutase (βPGM) catalyzes isomerization of β-Dglucose 1-phosphate (βG1P) into D-glucose 6-phosphate (G6P) via sequential phosphoryl transfer steps using a β-D-glucose 1,6-bisphosphate (βG16BP) intermediate. Synthetic fluoromethylenephosphonate and methylenephosphonate analogs of βG1P deliver novel step 1 transition state analog (TSA) complexes for βPGM, incorporating trifluoromagnesate and tetrafluoroaluminate surrogates of the phosphoryl group. Within an invariant protein conformation, the β-D-glucopyranose ring in the βG1P TSA complexes (step 1) is flipped over and shifted relative to the G6P TSA complexes (step 2). Its equatorial hydroxyl groups are hydrogen-bonded directly to the enzyme rather than indirectly via water molecules as in step 2. The (C)O-P bond orientation for binding the phosphate in the inert phosphate site differs by ∼30°between steps 1 and 2. By contrast, the orientations for the axial O-Mg-O alignment for the TSA of the phosphoryl group in the catalytic site differ by only ∼5°, and the atoms representing the five phosphorus-bonded oxygens in the two transition states (TSs) are virtually superimposable. The conformation of βG16BP in step 1 does not fit into the same invariant active site for step 2 by simple positional interchange of the phosphates: the TS alignment is achieved by conformational change of the hexose rather than the protein.phosphonate analogs | phosphoryl transfer mechanism | 19 F NMR | X-ray crystallography | water-mediated substrate recognition E fficient enzyme catalysis of the manipulation of phosphates is one of the great achievements of evolution (1). Enzymes that operate on phosphate monoesters and anhydrides transfer the phosphoryl moiety, PO 3 − , with rate accelerations approaching 10 21 for monoesters, placing them among the most proficient of all enzymes (1). Phosphomutases, including α-phosphoglucomutase (αPGM) (2, 3) and β-phosphoglucomutase (βPGM) (4-6), phosphoglycerate mutase (7), α-phosphomannomutase (αPMM/PGM) (8), and N-acetylglucosamine-phosphate mutase (9), merit special attention because these enzymes have to be effective in donating a phosphoryl group to either of two hydroxyl groups that have intrinsically different reactivity. Only when both half-reactions of a phosphomutase are accessible to mechanistic analysis can the problem of how an enzyme accommodates two distinct chemistries within a single active site be resolved. Hexose 1-phosphate mutases, including enzymes central to glycolysis and other metabolic pathways, are well characterized (10, 11). They are generally activated by phosphorylation to form a covalent phosphoenzyme, which then donates its PO 3 − group to either of its substrates to deliver a common, transient, hexose 1,6-bisphosphate intermediate species. However, structural studies on phosphomutases are complicated by the rapid and often imbalanced equilibrium position between the substrates, and kinetic studies are problematic because of competitive, parallel pathways of enzyme activation and substrate inhibition (12, ...

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“…57 The barrier for conversion of OMPDC and OMP to the transition state for enzyme-catalyzed decarboxylation [(Δ G ⧧ ) OMP ] is defined by the second-order rate constant ( k cat / K m ) OMP , while the barrier to formation of the transition state for OMPDC-catalyzed decarboxylation of the pieces EO + HP i [(Δ G ⧧ ) EO+HPi ] is defined by the third-order rate constant k cat / K HPi K EO (Scheme 6). Figure 5 presents the linear logarithmic free energy relationship between these barriers for wild-type and mutant OMPDC-catalyzed reactions of the whole substrate OMP and the substrate pieces EO + HP i .…”

Section: Discussionmentioning

confidence: 99%

Enzyme Architecture: Deconstruction of the Enzyme-Activating Phosphodianion Interactions of Orotidine 5′-Monophosphate Decarboxylase

et al. 2014

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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 kcat and Km 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-β-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 (kcat)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 (Km)obs.

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Enzyme Architecture: Remarkably Similar Transition States for Triosephosphate Isomerase-Catalyzed Reactions of the Whole Substrate and the Substrate in Pieces

Cited by 34 publications

References 41 publications

Reflections on the catalytic power of a TIM-barrel

Reflections on the catalytic power of a TIM-barrel

α-Fluorophosphonates reveal how a phosphomutase conserves transition state conformation over hexose recognition in its two-step reaction

Enzyme Architecture: Deconstruction of the Enzyme-Activating Phosphodianion Interactions of Orotidine 5′-Monophosphate Decarboxylase

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