Symmetrical Refolding of Protein Domains and Subunits: Example of the Dimeric Two-Domain 3-Isopropylmalate Dehydrogenases

Gráczer, Éva; Varga, Attila; Melnik, Bogdan S.; Semisotnov, Gennady V.; Závodszky, Péter; Vas, Mária

doi:10.1021/bi801857t

Cited by 7 publications

(13 citation statements)

References 53 publications

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“…This can explain the lower k cat values. In several other cases, tagged enzymes were used for kinetic measurements (49,50), which may have resulted in enzyme instability or interference with catalysis (51). We also found a few cases where enzymes were refolded from aggregated protein (52), suggesting that the measurement buffer might contain misfolded enzyme and, therefore, underestimate k cat .…”

Section: Discussionmentioning

confidence: 93%

Global characterization of in vivo enzyme catalytic rates and their correspondence to in vitro k _cat measurements

Davidi

Noor

Liebermeister

et al. 2016

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

242

382

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Turnover numbers, also known as k cat values, are fundamental properties of enzymes. However, k cat data are scarce and measured in vitro, thus may not faithfully represent the in vivo situation. A basic question that awaits elucidation is: how representative are k cat values for the maximal catalytic rates of enzymes in vivo? Here, we harness omics data to calculate k vivo max , the observed maximal catalytic rate of an enzyme inside cells. Comparison with k cat values from Escherichia coli, yields a correlation of r 2 = 0.62 in log scale (p < 10 −10 ), with a root mean square difference of 0.54 (3.5-fold in linear scale), indicating that in vivo and in vitro maximal rates generally concur. By accounting for the degree of saturation of enzymes and the backward flux dictated by thermodynamics, we further refine the correspondence between k vivo max and k cat values. The approach we present here characterizes the quantitative relationship between enzymatic catalysis in vitro and in vivo and offers a highthroughput method for extracting enzyme kinetic constants from omics data. (1-6). Many models of cellular metabolism include k cat values, the maximal turnover rates of enzymes, as key inputs to predict the behavior of metabolic pathways and networks (7-9). However, most values have never been measured experimentally. Escherichia coli is the most intensely biochemically characterized organism, but k cat values are available for only about 10% of its ≈ 2, 000 enzyme-reaction pairs (Dataset S1). Indeed, k cat values are missing for several central metabolic enzymes. The scarcity of kinetic data limits the scope of models and necessitates generic parameter assignments that significantly reduce the predictive power of cellular models.Even if a larger collection of k cat values was made available, their current use poses a major difficulty: k cat values are measured through in vitro enzyme assays, representing the initial rate of the reaction, i.e., full substrates saturation and negligible levels of products. Such assays may underrepresent factors like cellular metabolite concentrations, thermodynamic constraints, posttranslational modifications, chaperones, cellular crowding, and activating and inhibiting molecules, which can substantially alter enzyme kinetics in vivo. These omissions call into question the relevance of k cat measurements in vivo (10-12). Furthermore, an effort to measure a large number of k cat values under in vivo-like conditions presents a daunting challenge, given how many unknown biochemical factors might be involved.Several studies grapple with missing k cat values by sampling from the distribution of k cat values measured in vitro or by using measurements of the same enzyme from related species (13-16). These approximations systematically ignore any errors resulting from the differences between in vitro and in vivo environments. Approximations of this sort may also introduce significant errors, as k cat values can deviate by orders of magnitude between isozymes in the same organism as well a...

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Section: Discussionmentioning

confidence: 93%

Global characterization of in vivo enzyme catalytic rates and their correspondence to in vitro k _cat measurements

Davidi

Noor

Liebermeister

et al. 2016

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

242

382

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“…E270 of Tt ‐IPMDH was mutated into Ala using the QuickChange site‐directed mutagenesis kit. The modified enzyme was expressed and purified using the previously published method applied for the wild‐type enzyme [8]. The enzyme (about 30 mg/ml) was obtained in a 25 mM MOPS/KOH buffer, pH 7.6 and then lyophilised.…”

Section: Methodsmentioning

confidence: 99%

“…Abbreviations: IPMDH, 3-isopropylmalate dehydrogenase (EC 1.1.1.85); Tt, Thermus thermophilus; IPM, (2R,3S)-3-isopropylmalate; MOPS, 3-(N-morpholino)propane-sulphonic acid; SAXS, small angle X-ray scattering; FRET, fluorescence resonance energy transfer and purified using the previously published method applied for the wild-type enzyme [8]. The enzyme (about 30 mg/ml) was obtained in a 25 mM MOPS/KOH buffer, pH 7.6 and then lyophilised.…”

Section: Enzymes and Chemicalsmentioning

confidence: 99%

Glutamate 270 plays an essential role in K⁺‐activation and domain closure of Thermus thermophilus isopropylmalate dehydrogenase

et al. 2014

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Edited by Peter Brzezinski Keywords:Isopropylmalate dehydrogenase Activation by K + Site-directed mutagenesis X-ray crystallography Small angle X-ray scattering Fluorescence resonance energy transfer a b s t r a c tThe mutant E270A of Thermus thermophilus 3-isopropylmalate dehydrogenase exhibits largely reduced ($1%) catalytic activity and negligible activation by K + compared to the wild-type enzyme. A 3-4 kcal/mol increase in the activation energy of the catalysed reaction upon this mutation could also be predicted by QM/MM calculations. In the X-ray structure of the E270A mutant a water molecule was observed to take the place of K + . SAXS and FRET experiments revealed the essential role of E270 in stabilisation of the active domain-closed conformation of the enzyme. In addition, E270 seems to position K + into close proximity of the nicotinamide ring of NAD + and the electronwithdrawing effect of K + may help to polarise the aromatic ring in order to aid the hydride-transfer.

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“…From mutational studies with the Tt enzyme W77 and W195 could be identified as the participants in the FRET process. A reasonable structural explanation has been also provided: it was concluded that while the FRET signal of W77 reports on formation of the domain-closed conformation, that of W195 reports on the structural changes occurring at both subunit-subunit and domain-domain interfaces upon binding of IPM [8]. It is possible that the same type of mechanisms operate also in case of Ec enzyme where the equivalent side-chains are W81 and W205 [17].…”

Section: The Metal-ion Dependence Of the Fret Phenomenonmentioning

confidence: 95%

Essential role of the metal-ion in the IPM-assisted domain closure of 3-isopropylmalate dehydrogenase

Gráczer¹,

Konarev²,

Szimler³

et al. 2011

FEBS Letters

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Edited by Stuart FergusonKeywords: Domain movement Substrate and metal-effect 3-Isopropylmalate dehydrogenase Small-angle X-ray scattering a b s t r a c t X-ray structures of 3-isopropylmalate dehydrogenase (IPMDH) do not provide sufficient information on the role of the metal-ion in the metal-IPM assisted domain closure. Here solution studies were carried out to test its importance. Small-angle X-ray scattering (SAXS) experiments with the Thermus thermophilus enzyme (complexes with single substrates) have revealed only a very marginal (0-5%) extent of domain closure in the absence of the metal-ion. Only the metal-IPM complex, but neither the metal-ion nor the free IPM itself, is efficient in stabilizing the native protein conformation as confirmed by denaturation experiments with Escherichia coli IPMDH and by studies of the characteristic fluorescence resonance energy transfer (FRET) signal (from Trp to bound NADH) with both IPMDHs. A possible atomic level explanation of the metal-effect is given.

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Symmetrical Refolding of Protein Domains and Subunits: Example of the Dimeric Two-Domain 3-Isopropylmalate Dehydrogenases

Cited by 7 publications

References 53 publications

Global characterization of in vivo enzyme catalytic rates and their correspondence to in vitro k _cat measurements

Global characterization of in vivo enzyme catalytic rates and their correspondence to in vitro k _cat measurements

Glutamate 270 plays an essential role in K⁺‐activation and domain closure of Thermus thermophilus isopropylmalate dehydrogenase

Essential role of the metal-ion in the IPM-assisted domain closure of 3-isopropylmalate dehydrogenase

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Symmetrical Refolding of Protein Domains and Subunits: Example of the Dimeric Two-Domain 3-Isopropylmalate Dehydrogenases

Cited by 7 publications

References 53 publications

Global characterization of in vivo enzyme catalytic rates and their correspondence to in vitro k cat measurements

Global characterization of in vivo enzyme catalytic rates and their correspondence to in vitro k cat measurements

Glutamate 270 plays an essential role in K+‐activation and domain closure of Thermus thermophilus isopropylmalate dehydrogenase

Essential role of the metal-ion in the IPM-assisted domain closure of 3-isopropylmalate dehydrogenase

Contact Info

Product

Resources

About

Global characterization of in vivo enzyme catalytic rates and their correspondence to in vitro k _cat measurements

Global characterization of in vivo enzyme catalytic rates and their correspondence to in vitro k _cat measurements

Glutamate 270 plays an essential role in K⁺‐activation and domain closure of Thermus thermophilus isopropylmalate dehydrogenase