The Correlation of Reaction Kinetics and Substrate Binding with the Mechanism of Pyruvate Kinase

Reynard, A. M.; Hass, Louis F.; Jacobsen, D.D.; Boyer, Paul D.

doi:10.1016/s0021-9258(18)64071-2

Cited by 250 publications

(60 citation statements)

References 18 publications

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“…This preparation is also listed under 'lactate dehydrogenase' in the catalogue and is said to contain about equal specific activities ofthe two enzymes. The kinase is known to have no affinity for AMP (Reynard et al, 1961). Accordingly, on application of the enzyme mixture to an AMP-Sepharose column, pyruvate kinase activity appeared in the void volume completely free of measurable lactate dehydrogenase activity (Fig.…”

Section: Resultsmentioning

confidence: 99%

General ligands in affinity chromatography. Cofactor–substrate elution of enzymes bound to the immobilized nucleotides adenosine 5′-monophosphate and nicotinamide–adenine dinucleotide

Mosbach

Guilford

Ohlsson

et al. 1972

Biochemical Journal

201

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1. Two different gels have been prepared suitable for the separation of a number of enzymes, in particular NAD(+)-dependent dehydrogenases, by affinity chromatography. For both the matrix used was Sepharose 4B. For preparation (a), NAD(+)-Sepharose, 6-aminohexanoic acid has been coupled to the gel by the cyanogen bromide method and then NAD(+) was attached by using dicyclohexylcarbodi-imide; for preparation (b), AMP-Sepharose, N(6)-(6-aminohexyl)-AMP has been coupled directly to cyanogen bromide-activated gel. 2. Affinity columns of both gels retain only the two enzymes when a mixture of bovine serum albumin, lactate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase is applied. Subsequent elution with the cofactor NAD(+) yields glyceraldehyde 3-phosphate dehydrogenase whereas lactate dehydrogenase is eluted by applying the same molarity of the reduced cofactor. 3. The binding of both glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase to the gel tested, AMP-Sepharose, is strong enough to resist elution by gradients of KCl of up to at least 0.5m. A 0.0-0.15m gradient of the competitive inhibitor salicylate, however, elutes both enzymes efficiently and separately. 4. The elution efficiency of lactate dehydrogenase from AMP-Sepharose has been examined by using a series of eluents under comparable conditions of concentration etc. The approximate relative efficiencies are: 0 (lactate); 0 (lactate+semicarbazide); 0 (0.5mm-NAD(+)); 80 (lactate+NAD(+)); 95 (lactate+semicarbazide+NAD(+)); 100 (0.5mm-NADH). 5. All contaminating lactate dehydrogenase activity can be removed from commercially available crude pyruvate kinase in a single-step procedure by using AMP-Sepharose.

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Section: Resultsmentioning

confidence: 99%

General ligands in affinity chromatography. Cofactor–substrate elution of enzymes bound to the immobilized nucleotides adenosine 5′-monophosphate and nicotinamide–adenine dinucleotide

Mosbach

Guilford

Ohlsson

et al. 1972

Biochemical Journal

201

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“…ATPase activity was measured at 258C in a buffer that contained 30 mM KCl, 20 mM MOPS-KOH ( pH 7.5), 3 mM MgCl 2 , 2 mM ATP, 5 mM CaM, 40 unit ml 21 pyruvate kinase, 2.5 mM phosphoenolpyruvate, 1 mM EGTA or 0.2 mM CaCl 2 , 11-14 mg ml 21 M6 Full WT or the mutant, and various concentrations of F-actin ranging from 0 to 104 mM. The liberated pyruvate was determined as described previously [42]. To calculate the ATPase activity, protein concentrations of M6 Full WT and the mutant were determined using Coomassie Plus Assay Reagent (Thermo Scientific, Waltham, MA).…”

Section: Atpase Assaymentioning

confidence: 99%

The effect of novel mutations on the structure and enzymatic activity of unconventional myosins associated with autosomal dominant non-syndromic hearing loss

Kwon

Park³

et al. 2014

Open Biol.

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Mutations in five unconventional myosin genes have been associated with genetic hearing loss (HL). These genes encode the motor proteins myosin IA, IIIA, VI, VIIA and XVA. To date, most mutations in myosin genes have been found in the Caucasian population. In addition, only a few functional studies have been performed on the previously reported myosin mutations. We performed screening and functional studies for mutations in the MYO1A and MYO6 genes in Korean cases of autosomal dominant non-syndromic HL. We identified four novel heterozygous mutations in MYO6. Three mutations (p.R825X, p.R991X and Q918fsX941) produce a premature truncation of the myosin VI protein. Another mutation, p.R205Q, was associated with diminished actin-activated ATPase activity and actin gliding velocity of myosin VI in an in vitro analysis. This finding is consistent with the results of protein modelling studies and corroborates the pathogenicity of this mutation in the MYO6 gene. One missense variant, p.R544W, was found in the MYO1A gene, and in silico analysis suggested that this variant has deleterious effects on protein function. This finding is consistent with the results of protein modelling studies and corroborates the pathogenic effect of this mutation in the MYO6 gene.

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“…This conclusively shows that the enzyme is inhibited by the product ATP but not by pyruvate, in agreement with previous studies. 39,48 This makes sense, as PEP binding is largely driven by the phosphate group, which is absent from pyruvate. 41,49 Since enzyme activity is not affected by pyruvate, it will be ignored in what follows and the reaction will be discussed in terms of [PEP], [ADP], and [ATP].…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…Thus, the random sequential model clearly matches the experimental data better than either the ping-pong or ordered sequential mechanism, in agreement with the reported mechanism for this enzyme. 37,39 Furthermore, the large value of K ii ATP indicates that the dead-end complex rMPK−PEP−ATP does not readily form. Thus, eq 8 can be simplified to…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Characterizing Bi-substrate Enzyme Kinetics at High Resolution by 2D-ITC

Wang

Mittermaier

2021

Anal. Chem.

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There is growing interest in using isothermal titration calorimetry (ITC) to characterize enzyme kinetics by measuring the heat produced or absorbed by catalysis in real time. Since virtually all chemical reactions are associated with changes in enthalpy, ITC represents a robust and nearly universal experimental approach. Nevertheless, there are technical challenges that limit ITC’s applicability. For instance, the full kinetic characterization of enzymes with two substrates (bi-substrate enzymes), which comprise the majority of known examples, requires a series of experiments to be performed as the concentrations of both substrates are varied. This is a time-consuming and expensive process using current ITC methods since many (>5) individual experiments must be performed independently to obtain a sufficient quantity of data. We have developed a new ITC method, which we term 2D-ITC, which maps the reaction velocity as a function of two substrate concentrations in a single, roughly 2 h long experiment. This method provides a level of detail that rivals or exceeds any existing enzyme assay, as a single experiment generates on the order of 7000 catalytic rate measurements. In a proof-of-principle application to rabbit muscle pyruvate kinase (rMPK), the method correctly identified the enzyme’s random sequential mechanism and allosteric catalytic suppression by the amino acid phenylalanine (Phe). Unexpectedly, we found that while Phe reduces affinity for the substrate phosphoenolpyruvate, a known phenomenon, it also alleviates inhibition by the reaction product ATP, which had not been reported previously. Given the relative abundance of ATP in the cell, this opposing effect is expected to have a substantial impact on rMPK activity.

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The Correlation of Reaction Kinetics and Substrate Binding with the Mechanism of Pyruvate Kinase

Cited by 250 publications

References 18 publications

General ligands in affinity chromatography. Cofactor–substrate elution of enzymes bound to the immobilized nucleotides adenosine 5′-monophosphate and nicotinamide–adenine dinucleotide

General ligands in affinity chromatography. Cofactor–substrate elution of enzymes bound to the immobilized nucleotides adenosine 5′-monophosphate and nicotinamide–adenine dinucleotide

The effect of novel mutations on the structure and enzymatic activity of unconventional myosins associated with autosomal dominant non-syndromic hearing loss

Characterizing Bi-substrate Enzyme Kinetics at High Resolution by 2D-ITC

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