Internal thermodynamics of enzymes determined by equilibrium quench: values of Kint for enolase and creatine kinase

Burbaum, Jonathan J.; Knowles, Jeremy R.

doi:10.1021/bi00450a010

Cited by 45 publications

(34 citation statements)

References 64 publications

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“…At high PGA concentrations the symmetric complex (hNSE•Mg 2 •PGA) 2 is initially observed, which is consistent with kinetic data showing that both subunits are simultaneously active [31]. Solution studies [32, 33] have shown that upon reaching equilibrium, the ratio of PGA/PEP bound to enolase is 1:1 and this is consistent with the observation of the asymmetric dimer (hNSE•2Mg 2+ •PGA)(hNSE•2Mg 2+ •PEP) after 30 days of crystal soaking, when presumably the equilibrium was approached.…”

Section: Discussionsupporting

confidence: 78%

Structures of asymmetric complexes of human neuron specific enolase with resolved substrate and product and an analogous complex with two inhibitors indicate subunit interaction and inhibitor cooperativity

Qin

Chai

Brewer

et al. 2012

Journal of Inorganic Biochemistry

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In the presence of magnesium, enolase catalyzes the dehydration of 2-phospho-D-glycerate (PGA) to phosphoenolpyruvate (PEP) in glycolysis and the reverse reaction in gluconeogensis at comparable rates. The structure of human neuron specific enolase (hNSE) crystals soaked in PGA showed that the enzyme is active in the crystals and produced PEP; conversely soaking in PEP produced PGA. Moreover, the hNSE dimer contains PGA bound in one subunit and PEP or a mixture of PEP and PGA in the other. Crystals soaked in a mixture of competitive inhibitors tartronate semialdehyde phosphate (TSP) and lactic acid phosphate (LAP) showed asymmetry with TSP binding in the same site as PGA and LAP in the PEP site. Kinetic studies showed that the inhibition of NSE by mixtures of TSP and LAP is stronger than predicted for independently acting inhibitors. This indicates that in some cases inhibition of homodimeric enzymes by mixtures of inhibitors (“heteroinhibition”) may offer advantages over single inhibitors.

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Section: Discussionsupporting

confidence: 78%

Structures of asymmetric complexes of human neuron specific enolase with resolved substrate and product and an analogous complex with two inhibitors indicate subunit interaction and inhibitor cooperativity

Qin

Chai

Brewer

et al. 2012

Journal of Inorganic Biochemistry

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“…No enzyme would be expected to have significantly approached the ultimate state of catalytic perfection where the optimal K,,, value is predicted necessarily to become close to unity, but one might expect to find a general correlation between experimentally observed K,,, values and the magnitude of the overall equilibrium constant. Examination of the list of enzyme reaction compiled by Burbaum and Knowles [14] suggests that such a correlation indeed exists with regard to the few systems listed that may actually be assumed to operate by the one-substrate mechanism in Scheme 1 . This is illustrated by the data shown in Table 2 and lends support to the supposition of Benner and collaborators [15, 161 that K,,, for an enzymically catalysed reaction should reflect the magnitude of the overall equilibrium constant rather than being equal to unity.…”

Section: Triosephosphate Isomerasementioning

confidence: 98%

Why do many Michaelian enzymes exhibit an equilibrium constant close to unity for the interconversion of enzyme‐bound substrate and product?

Pettersson¹

1991

European Journal of Biochemistry

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1. The idea is advanced that an evolutionary pressure in the direction of increased metabolic fluxes should lead to the selection at any evolutionary stage of enzymes which are optimally efficient considering the total evolutionary effort which has been spent on their improvement as catalysts. Relationships are derived which may be used for determination of the optimal state of operation of a Michaelian enzyme at a given total evolutionary effort as measured, for instance, by the mean magnitude of variable rate constants in the reaction mechanism. These relationships are applied to characterize the dependence on the total evolutionary effort of the optimal value of the equilibrium constant (Kin,) for the conversion of enzyme-bound substrate into enzyme-bound product.2. The results show that the optimal value of Kin, reflects primarily the magnitude of the overall equilibrium constant for the catalysed reaction, whether or not the enzyme operates close to equilibrium. When the total evolutionary effort tends towards infinitely high values, Kin, approaches unity, regardless of the magnitude of the overall equilibrium constant and of the concentrations of substrate and product. Concomitantly, the enzyme approaches its ultimate state of catalytic perfection where all variable rate constants in the mechanism tend towards infinitely high values.3. It is concluded from previously reported kinetic data that triosephosphate isomerase has been subjected to such a low total evolutionary effort that the enzyme cannot possibly have reached (or even significantly approached) the ultimate state of perfection where the Kin, value necessarily becomes close to unity. The reason why this enzyme exhibits a Kin, value relatively close to unity is that it catalyses a reaction with an equilibrium constant relatively close to unity. Evidence is presented to show that similar considerations may apply for other enzymes operating by the examined reaction mechanism.4. Analytical data are reported which indicate that Kin, ultimately approaches unity because forward and reverse rate constants for the interconversion of enzyme-bound substrate and product are equivalent with regard to the evolutionary effort required to increase their magnitude.The evolutionary development of enzymes may certainly be dependent on a multitude of factors relating to their function as biologically regulated catalysts affecting both metabolite levels and reaction fluxes in metabolic pathways. Since enzymes serve the obvious primary purpose of maintaining high reaction fluxes, however, they can be anticipated to have evolved primarily in response to a selective pressure tending to increase their catalytic efficiency. Fundamental functional properties of presently existing enzymes, therefore, may be difficult to explain unless we have a thorough knowledge of the likely effects of such a selective pressure on the catalytic reaction mechanism. In particular, it would seem important to know how enzymic reaction mechanisms should be kinetically designed in order to opti...

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“…The occupancy of the wild-type subunit is consistent with the internal thermodynamics of enolase in solution. 12 Figure 5 shows the F o −F c density map for the substrate in the K345A subunit. The presence of the hydroxyl group of 2-PGA is clearly defined.…”

Section: Crystal Structuresmentioning

confidence: 99%

Structure and Catalytic Properties of an Engineered Heterodimer of Enolase Composed of One Active and One Inactive Subunit

Sims¹,

Menefee²,

Larsen³

et al. 2006

Journal of Molecular Biology

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Enolase is a dimeric enzyme that catalyzes the interconversion of 2-phospho-D-glycerate and phosphoenolpyruvate. This reversible dehydration is effected by general acid-base catalysis that involves, principally, Lys345 and Glu211 (numbering system of enolase 1 from yeast). The crystal structure of the inactive E211Q enolase shows that the protein is properly folded. However, K345 variants have, thus far, failed to crystallize. This problem was solved by crystallization of an engineered heterodimer of enolase. The heterodimer was composed of an inactive subunit that has a K345A mutation and an active subunit that has N80D and N126D surface mutations to facilitate ion-exchange chromatographic separation of the three dimeric species. The structure of this heterodimeric variant, in complex with substrate/product, was obtained at 1.85 Å resolution. The structure was compared to a new structure of wild-type enolase obtained from crystals belonging to the same space group. Asymmetric dimers having one subunit exhibiting two of the three active site loops in an open conformation and the other in a conformation having all three loops closed appear in both structures. The K345A subunit of the heterodimer is in the loop-closed conformation; its C α carbon atoms closely match those of the corresponding subunit of wild-type enolase (root-mean-squared deviation of 0.23 Å). The k cat and k cat /K m values of the heterodimer are approximately half those of the N80D/N126D homodimer, which suggests that the subunits in solution are kinetically independent. A comparison of enolase structures obtained from crystals belonging to different space groups suggests that asymmetric dimers can be a consequence of the asymmetric positioning of the subunits within the crystal lattice.

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Internal thermodynamics of enzymes determined by equilibrium quench: values of Kint for enolase and creatine kinase

Cited by 45 publications

References 64 publications

Structures of asymmetric complexes of human neuron specific enolase with resolved substrate and product and an analogous complex with two inhibitors indicate subunit interaction and inhibitor cooperativity

Structures of asymmetric complexes of human neuron specific enolase with resolved substrate and product and an analogous complex with two inhibitors indicate subunit interaction and inhibitor cooperativity

Why do many Michaelian enzymes exhibit an equilibrium constant close to unity for the interconversion of enzyme‐bound substrate and product?

Structure and Catalytic Properties of an Engineered Heterodimer of Enolase Composed of One Active and One Inactive Subunit

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