pH-dependence of the triose phosphate isomerase reaction

Plaut, Barbara; Knowles, Jeremy R.

doi:10.1042/bj1290311

Cited by 162 publications

(160 citation statements)

References 27 publications

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“…We obtained similar results with chicken muscle triosephosphate isomerase which renatured within 15 s after direct dilution of a solution of the enzyme in 6 M guanidinium chloride to a final denaturant concentration ofO.1 M. Plaut and Knowles [8] have shown that incubation of chicken muscle triosephosphate isomerase in various buffers at 38 -C causes an irreversible loss of enzymic activity under conditions removed from the pH optimum (pH 6.8 -7.8) of the enzyme activity. Fig.…”

Section: Determination Of the Optiniurn Conditions Jor Renaturationsupporting

confidence: 72%

The Denaturation‐Renaturation of Chicken‐Muscle Triosephosphate Isomerase in Guanidinium Chloride

McVITTIE

Esnouf

Peacocke

1977

European Journal of Biochemistry

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1 . The process of denaturation of the chicken muscle dimeric enzyme triosephosphate isomerase on addition of guanidinium chloride has been studied at pfI 7.6, the pH at which the recovery of activity is optimal (100 %) on removal of denaturant. Determinations of the sedimentation coefficient, intrinsic viscosity, molecular weight (by sedimentation equilibrium studies) and the absorption coefficient at 280 nm in various concentrations of guanidinium chloride concurred in showing a single, sharp transition at about 0.7 M guanidinium chloride at a protein concentration 1 -5 mg/ml from the native enzyme to the dissociated, unfolded chains of the monomer. Relative fluorescent intensity measurements revealed a single transition at about 0.4 M guanidinium chloride at enzyme concentrations of about 0.05 mg/ml.2. The process of denaturation in different guanidinium chloride concentrations was first order with respect to enzyme and about sixth order with respect to denaturant.3. The rate of attainment of equilibrium during the renaturation obeyed second-order/first-order reversible kinetics. It was concluded that the rate-determining step in renaturation at pH 7.6 must be the association of two subunits.It has long been recognised that the three-dimensional structure of a protein (or enzyme) is a function of, amongst other factors, the amino-acid sequence of that protein [l -31. Although the cellular apparatus of protein synthesis involves the presence of ribosomes, substrates and other effectors, the process of protein folding from a fully random conformation has been mimicked many times in cell-free systems in vitro. Often, such processes appear to involve, as intermediates, structures other than the fully unfolded and folded, native, conformations and the idea of pathways of protein folding involving 'nucleation' centres in polypeptide chains is now widely accepted. To date, however, the bulk of the work has involved relatively small proteins composed of a single subunit. There has been comparitively little study of multi-subunit enzymes where, as well as chain folding, inter-chain association must be important in determining the final quaternary structure of the protein.In a previous paper [4] we have shown that the dimeric enzyme triosephosphate isomerase unfolds and dissociates simultaneously in guanidinium chloride solutions. Waley [5] demonstrated that, upon removal of the denaturant, enzymic activity can be regained.

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Section: Determination Of the Optiniurn Conditions Jor Renaturationsupporting

confidence: 72%

The Denaturation‐Renaturation of Chicken‐Muscle Triosephosphate Isomerase in Guanidinium Chloride

McVITTIE

Esnouf

Peacocke

1977

European Journal of Biochemistry

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“…The apparent pK a values of the ionizable groups in the wild-type yTIM 33 are 6.05 and 9.05, whereas they are around 6.6 and >9.0 respectively in the NovoTIM designs. It has been shown that distributed mutations can contribute significantly to the tuning of pK a values.…”

Section: Discussionmentioning

confidence: 90%

“…50 Triose phosphate isomerase activity was measured under steady-state conditions in the DHAP→GAP direction, using a coupled enzyme assay that links GAP production to reduction of NAD +. 33 Buffers, salts and DHAP (sodium salt) were purchased from Sigma-Aldrich; enzymes and NAD + for the enzyme-linked assay were purchased from Roche. The inhibitor phosphoglycolohydroxamate was custom synthesized by Gateway chemicals.…”

Section: Methodsmentioning

confidence: 99%

RETRACTED: Local Encoding of Computationally Designed Enzyme Activity

Allert

Dwyer

Hellinga

2007

Journal of Molecular Biology

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One aim of computational protein design is to introduce novel enzyme activity into proteins of known structure by predicting mutations that stabilize transition states. Previously we have shown that it is possible to introduce triose phosphate isomerase activity into the ribose-binding protein of Escherichia coli by constructing 17 mutations in the first two layers of residues that surround the wild-type ligand-binding site. Here we report that these mutations can be "transplanted" into a homologous ribose-binding protein, isolated from the hyperthermophilic bacterium Thermoanaerobacter tengcongensis, with retention of catalytic activity, substrate affinity, and reaction pH dependence. The observed 10 5 -10 6 -fold rate enhancement corresponds to 70% of the maximally known transition-state binding energy. The wild-type sequences in these two homologues are almost perfectly conserved in the vicinity of their ribose-binding sites, but diverge significantly at increasing distance from these sites. The results demonstrate that the computationally designed mutations are sufficient to encode the observed enzyme activity, that all the observed activity is locally encoded within the layer of residues directly in contact with the substrate, and that in this case at least 70% of transition state stabilization energy can be achieved using straightforward considerations of stereochemical complementarity between enzyme and reactants.

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“…The kinetic parameters of the mutant are essentially the same as those for the wild-type enzyme (27). Enzymatic activity was determined by the conversion of GAP to DHAP in the presence of TIM using an assay linked to glycerol 3-phosphate dehydrogenase as previously described (45).…”

Section: Methodsmentioning

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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pH-dependence of the triose phosphate isomerase reaction

Cited by 162 publications

References 27 publications

The Denaturation‐Renaturation of Chicken‐Muscle Triosephosphate Isomerase in Guanidinium Chloride

The Denaturation‐Renaturation of Chicken‐Muscle Triosephosphate Isomerase in Guanidinium Chloride

RETRACTED: Local Encoding of Computationally Designed Enzyme Activity

Substrate product equilibrium on a reversible enzyme, triosephosphate isomerase

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