Dihydroxyacetone phosphate. Its structure and reactivity with α-glycerophosphate dehydrogenase, aldolase and triose phosphate isomerase and some possible metabolic implications

Reynolds, Sarah J.; Yates, David; Pogson, C. I.

doi:10.1042/bj1220285

Cited by 126 publications

(119 citation statements)

References 34 publications

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“…The solid line shows the fit of the data to the Michaelis-Menten equation with values of (K m ) app = 0.24 mM and V max = 1.42×10 −7 M s −1 . The latter was used to calculate k cat = 130 s −1 , and a value of K m = 0.13 mM for the reactive free carbonyl form of DHAP was calculated using (1 -f hyd ) = 0.55 for the fraction of DHAP present in the free carbonyl form (Scheme 3) (17). These data (Table 1) are in agreement with the published kinetic parameters for GPDH (18).…”

Section: Resultssupporting

confidence: 79%

A Substrate in Pieces: Allosteric Activation of Glycerol 3-Phosphate Dehydrogenase (NAD⁺) by Phosphite Dianion

2008

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The ratio of the second-order rate constants for reduction of dihydroxyacetone phosphate (DHAP) and of the neutral truncated substrate glycolaldehyde (GLY) by glycerol 3-phosphate dehydrogenase (NAD + , GPDH) saturated by NADH is (1.0×10 6 M −1 s −1 )/(8.7×10 −3 M −1 s −1 ) = 1.1×10 8 , which was used to calculate an intrinsic phosphate binding energy of ≤ −11.0 kcal/mol. Phosphite dianion binds very weakly to GPDH (K d > 0.1 M), but the bound dianion strongly activates GLY towards enzymecatalyzed reduction by NADH. Thus, the large intrinsic phosphite binding energy is expressed only at the transition state for the GPDH-catalyzed reaction. The ratio of rate constants for the phosphite-activated and the unactivated GPDH-catalyzed reduction of GLY by NADH is (4300 M −2 s −1 )/(8.7×10 −3 M −1 s −1 ) = 5×10 5 M −1 , which was used to calculate an intrinsic phosphite binding energy of −7.7 kcal/mol for the association of phosphite dianion with the transition state complex for the GPDH-catalyzed reduction of GLY. Phosphite dianion has now been shown to activate bound substrates for enzyme-catalyzed proton transfer, decarboxylation, hydride transfer and phosphoryl transfer reactions. Structural data provides strong evidence that enzymic activation by the binding of phosphite dianion occurs at a modular active site featuring: (1) a binding pocket complementary to the reactive substrate fragment, and which contains all the active site residues needed to catalyze the reaction of the substrate piece or of the whole substrate; (2) a phosphate/phosphite dianion binding pocket that is completed by the movement of flexible protein loop(s) to surround the nonreacting oxydianion. We propose that loop motion and associated protein conformational changes that accompany the binding of phosphite dianion and/or phosphodianion substrates leads to encapsulation of the substrate and/or its pieces in the protein interior, and to placement of the active site residues in positions where they provide optimal stabilization of the transition state for the catalyzed reaction.Enzymes are protein catalysts that effect much larger rate accelerations than those observed for small molecule catalysts. The larger rate accelerations for enzymes are a consequence of the greater binding affinities of enzymes for their reaction transition states (1). Enzyme catalysis is so efficient that the release of products would be intolerably slow if they were to bind with the same affinity as the transition state. For example, the ca. 30 kcal/mol transition state intrinsic binding energy estimated for the enzymatic decarboxylation of orotidine 5′-monophosphate (OMP) 1 catalyzed by orotidine 5′-monophosphate decarboxylase (OMPDC) (2) is even larger than the 20 kcal/mol binding energy associated with the effectively irreversible binding of biotin to avidin (3). Consequently, in order to avoid this free energy "trap", enzymes generally bind their substrates/products much more weakly than the reaction transition state.

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Section: Resultssupporting

confidence: 79%

A Substrate in Pieces: Allosteric Activation of Glycerol 3-Phosphate Dehydrogenase (NAD⁺) by Phosphite Dianion

2008

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“…Because of the high selectivity of TIM's active site, the hydrated forms bind weakly as competitive inhibitors (23). The DHAP hydrate/keto ratio is 45:55 at 20°C, pH 7.5, with a conversion rate of k hydration ϭ 3.6 ϫ 10 Ϫ1 ⅐s Ϫ1 and k dehydration 4.4 ϫ 10 Ϫ1 ⅐s Ϫ1 (38). For GAP, the hydrate/ aldehyde ratio is 29:1, with a conversion rate of 8.7 ϫ 10 Ϫ2 ⅐s Ϫ1 in the pH range 7.3-8.6, at 20°C (37).…”

Section: Resultsmentioning

confidence: 99%

“…Enzymatic assays (37) have established that the free aldehyde is produced by the enzyme in the biologically relevant, endergonic reaction from DHAP to GAP. Similarly, in the exergonic direction from GAP to DHAP, the enzyme produces the free keto form of DHAP (38). Because of the high selectivity of TIM's active site, the hydrated forms bind weakly as competitive inhibitors (23).…”

Section: Resultsmentioning

confidence: 99%

Substrate product equilibrium on a reversible enzyme, triosephosphate isomerase

Rozovsky

McDermott

2007

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

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The highly efficient glycolytic enzyme, triosephosphate isomerase, is expected to differentially stabilize the proposed stable reaction species: ketone, aldehyde, and enediol(ate). The identity and steady-state populations of the chemical entities bound to triosephosphate isomerase have been probed by using solid-and solution-state NMR. The 13 C-enriched ketone substrate, dihydroxyacetone phosphate, was bound to the enzyme and characterized at steady state over a range of sample conditions. The ketone substrate was observed to be the major species over a temperature range from ؊60°C to 15°C. Thus, there is no suggestion that the enzyme preferentially stabilizes the reactive intermediate or the product. The predominance of dihydroxyacetone phosphate on the enzyme would support a mechanism in which the initial proton abstraction in the reaction from dihydroxyacetone phosphate to D-glyceraldehyde 3-phosphate is significantly slower than the subsequent chemical steps.enzymatic catalysis ͉ Michaelis complex ͉ solid-state NMR T he glycolytic enzyme triosephosphate isomerase (TIM) catalyzes isomerization of a ketone to an aldehyde, progressing via two successive proton transfers involving carbon acids and additional proton transfers for the attached oxygen atoms (Fig. 1). The precise path of the transferred proton and the identity of the reaction intermediates have been, by and large, deduced from the fate of isotopic labels (1, 2). The existence of a stable enediol(ate) intermediate has been inferred from the loss of an isotope label from the substrate during the reaction (1, 3); this reaction intermediate must exist with a sufficient lifetime to allow partial equilibration of the catalytic base with the surrounding solvent. Moreover, when the reaction takes place in tritiated water the solvent's tritium is incorporated into both the substrate and product, evidence that the hydrogen of the newly formed carbonhydrogen bond is mostly derived from the solvent (4, 5) and that exchange occurs from an enzyme bound intermediate. The stereochemistry of the reaction (2) and the orientation of the substrate in the Michaelis complex (6) strongly suggest that the reaction intermediate is a cis-enediol(ate). Based on a comprehensive study of isotope effects supplemented by extensive biophysical investigations of the reaction mechanism (7, 8), Albery and Knowles (9) constructed a kinetically detailed energy profile for the reversible interconversion of substrate, product, and one intermediate subject to isotope exchange with solvent ( Fig. 1).This now well known reaction energy profile illustrated for the first time many of the essential elements contributing to the high catalytic efficiency brought about by enzymes (10). For example, the free-energy profile illustrates the potential catalytic power of preferential stabilization of intermediates in a reaction. The high catalytic efficiency of TIM [almost 10 orders of magnitude compared with the reaction catalyzed by an acetate ion (11)] is thought to arise from matching the pK a va...

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“…Fractions were assayed for enzyme activity, and samples of the peak fractions were further analyzed by NaDod-S04/polyacrylamide electrophoresis on a 10% gel (26), after which protein was visualized with Kodavue stain (Eastman Kodak). The fractions that contained the mutant isomerase (fractions [32][33][34][35][36][37][38][39][40][41][42][43][44] were pooled and concentrated by dialysis against 20 mM Tris HCI, pH 7.3/1 mM EDTA/polyethylene glycol [Mr, >20,000; 10% (wt/vol)] at 40C for 48 hr. The final volume after dialysis was 1 ml, and the protein concentration was 0.46 mg/ml.…”

Section: Methodsmentioning

confidence: 99%

Active site of triosephosphate isomerase: in vitro mutagenesis and characterization of an altered enzyme.

Straus¹,

Raines²,

Kawashima³

et al. 1985

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

105

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We have replaced the glutamic acid-165 at the active site of chicken triosephosphate isomerase with an aspartic acid residue using site-directed mutagenesis. Expression of the mutant protein in a strain of Escherichia coil that lacks the bacterial isomerase results in a complementation phenotype that is intermediate between strains that have no isomerase and strains that produce either the wild-type chicken enzyme or the native E. coli isomerase. The value of kct for the purified mutant enzyme when glyceraldehyde 3-phosphate is the substrate is 1/1500th that of the wild-type enzyme, and the Km is decreased by a factor of 3.6. With dihydroxyacetone phosphate as substrate, the k value is 1/240th that of the wild-type enzyme, and Km is 2 times higher. The value ofK1 for a competitive inhibitor, phosphoglycolate, is the same for the mutant and wild-type enzymes, at 2 x 10`5 M. By treating the enzyme-catalyzed isomerization as a simple three step process and assuming that substrate binding is diffusion limite", it is evident that the mutation of glutamic acid-165 to aspartic acid principally affects the free energy of the transition state~s) for the catalytic reaction itself.Triosephosphate isomerase (TIM; D-glyceraldehyde 3-phosphate ketol-isomerase, EC 5.3.1.1) is a glycolytic enzyme that interconverts dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP) (Fig. 1). The second-order rate constant for the reaction in the downhill direction (GAP -* DHAP) is 101o times faster for TIM catalysis than for acetate ion catalysis, and it is close to the theoretical limit for the diffusive encounter of substrate and enzyme (1, 2).Kinetic and mechanistic analyses of the TIM-catalyzed reaction have led to a plausible pathway for this transformation ( Fig. 1). There is good evidence that the isomerization proceeds through a cis-enediol (or enediolate) intermediate (3). B-in Fig. 1 represents a base, almost certainly the carboxylate group of glutamic acid-165, which abstracts the pro-R hydrogen from substrate DHAP. The conjugate acid of this group (BH in Fig. 1 Fig. 1) is thought to stabilize the developing negative charges on the oxygens at C-1 and C-2 during formation of the enediol(ate), polarizing the carbonyl group in the enzyme-substrate complex and facilitating the C-H bond cleavage reactions (7-9). From measurements of the fate of isotopic labels in the substrate and in solvent, Albery and Knowles were able to determine the rate constants for each step of the reaction and to construct a Gibbs free energy profile for the catalyzed process (10).

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Dihydroxyacetone phosphate. Its structure and reactivity with α-glycerophosphate dehydrogenase, aldolase and triose phosphate isomerase and some possible metabolic implications

Cited by 126 publications

References 34 publications

A Substrate in Pieces: Allosteric Activation of Glycerol 3-Phosphate Dehydrogenase (NAD⁺) by Phosphite Dianion

A Substrate in Pieces: Allosteric Activation of Glycerol 3-Phosphate Dehydrogenase (NAD⁺) by Phosphite Dianion

Substrate product equilibrium on a reversible enzyme, triosephosphate isomerase

Active site of triosephosphate isomerase: in vitro mutagenesis and characterization of an altered enzyme.

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Dihydroxyacetone phosphate. Its structure and reactivity with α-glycerophosphate dehydrogenase, aldolase and triose phosphate isomerase and some possible metabolic implications

Cited by 126 publications

References 34 publications

A Substrate in Pieces: Allosteric Activation of Glycerol 3-Phosphate Dehydrogenase (NAD+) by Phosphite Dianion

A Substrate in Pieces: Allosteric Activation of Glycerol 3-Phosphate Dehydrogenase (NAD+) by Phosphite Dianion

Substrate product equilibrium on a reversible enzyme, triosephosphate isomerase

Active site of triosephosphate isomerase: in vitro mutagenesis and characterization of an altered enzyme.

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A Substrate in Pieces: Allosteric Activation of Glycerol 3-Phosphate Dehydrogenase (NAD⁺) by Phosphite Dianion

A Substrate in Pieces: Allosteric Activation of Glycerol 3-Phosphate Dehydrogenase (NAD⁺) by Phosphite Dianion