The determination of thermodynamic allosteric parameters of an enzyme undergoing steady-state turnover

Reinhart, Gregory D.

doi:10.1016/0003-9861(83)90225-4

Cited by 84 publications

(126 citation statements)

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“…The coupling constant Q ax describes the nature and magnitude of the allosteric response (10,11). Q ax will be greater than 1 when MgADP acts as an activator of Fru-6-P binding, and Q ax is less than 1 when MgADP causes inhibition of Fru-6-P binding.…”

Section: Methodsmentioning

confidence: 99%

Kinetic and Structural Characterization of Phosphofructokinase from Lactobacillus bulgaricus

Paricharttanakul

Menefee

et al. 2005

Biochemistry

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Phosphofructokinase from Lactobacillus delbrueckii subspecies bulgaricus (LbPFK) has been reported to be a nonallosteric analogue of phosphofructokinase from Escherichia coli at pH 8.2 [Le Bras et al. (1991) Eur. J. Biochem. 198, 683-687]. A reexamination of the kinetics of this enzyme shows LbPFK to have limited binding affinity toward the allosteric ligands, MgADP and PEP, with dissociation constants of approximately 20 mM for both. Their allosteric effects are observed only at high concentrations of these ligands, with both exhibiting inhibitory effects on substrate binding. No pH dependence was observed for the binding and the influence of MgADP and PEP on the enzyme. To attempt to explain these results, the crystal structure of LbPFK was solved using molecular replacement to 1.86 A resolution. A comparative study of the LbPFK structure with that of phosphofructokinases from E. coli (EcPFK) and Bacillus stearothermophilus (BsPFK) reveals a structure with conserved fold and substrate binding site. The effector binding site, however, shows many differences that could explain the observed decreases in binding affinity for MgADP and PEP in LbPFK as compared to the other two enzymes.

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Section: Methodsmentioning

confidence: 99%

Kinetic and Structural Characterization of Phosphofructokinase from Lactobacillus bulgaricus

Paricharttanakul

Menefee

et al. 2005

Biochemistry

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“…Provided aspartate achieves rapid binding equilibrium in the steady state, the effect that an allosteric ligand has on [Asp] 0.5 is quantitatively described by the parameter Q ax , which is defined as: (Reinhart, 1983). The nature of the allosteric effector is indicated by the value of ΔG ax :…”

Section: Discussionmentioning

confidence: 99%

Temperature effects on the allosteric responses of native and chimeric aspartate transcarbamoylases 1 1Edited by J. H. Miller

Liu

Wales

Wild

1998

Journal of Molecular Biology

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Although structurally very similar, the aspartate transcarbamoylases (ATCase) of Serratia marcescens and Escherichia coli have distinct allosteric regulatory patterns. It has been reported that a S. marcescens chimera, SM : rS5′ec, in which five divergent residues (r93 to r97) of the regulatory polypeptide were replaced with their Escherichia coli counterparts, possessed E. colilike regulatory characteristics. T he reverse chimera EC : rS5′sm, in which the same five residues of E. coli have been replaced with their S. marcescens counterpart, lost both heterotrophic and homotropic responses. These results indicate that the r93-r97 region is critical in defining the ATCase allosteric character. Molecular modeling of the regulatory polypeptides has suggested that the replacement of the S5′ β-strand resulted in disruption of the allosteric-zinc interface. However, the structure-function relationship could be indirect, and the disruption of the interface could influence allostery by altering the global energy of the enzyme. Studies of the temperaturesensitivity of the CTP response demonstrate that it is possible to convert CTP inhibition of the SM : rS5′ec chimera at high temperature to activation below 10 °C. Nonetheless, the temperature response of the native S. marcescens ATCase suggests a strong entropic effect that counteracts the CTP activation. Therefore, it is suggested that the entropy component of the coupling free energy plays a significant role in the determination of both the nature and magnitude of the allosteric effect in ATCase.

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“…A full, linked-function determination of Q ax and W ax involves monitoring the €dependence of each rate parameter on a broad range of concentrations of allosteric ligand to experimentally establish the limiting ratios (15,31). Here, however, given the large number of CPS variants as well the multiplicity of reaction types, values of the coupling parameters have been approximated by measuring K m and V max only in the absence and presence of saturating effector.…”

Section: Evaluation Of Allosteric Coupling Interactionsmentioning

confidence: 99%

Resolving the Fluorescence Response of Escherichia coli Carbamoyl Phosphate Synthetase: Mapping Intra- and Intersubunit Conformational Changes

et al. 2006

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Carbamoyl phosphate synthetase (CPS) from E. coli is potentially overlaid with a network of allosterism, interconnecting active sites, effector binding sites, and aggregate interfaces to control its mechanisms of catalytic synchronization, regulation, and oligomerization, respectively. To characterize these conformational changes, a tryptophan-free variant of CPS was genetically engineered by substituting six native tryptophans with tyrosines. Each tryptophan was then reinserted, singly, as a specific fluorescence probe of its corresponding microenvironment. The amino acid substitutions themselves result in little apparent disruption of the protein; variants maintain catalytic and allosteric functionality, and the fluorescence properties of each tryptophan, while unique, are additive to wild-type CPS. Whereas the collective, intrinsic fluorescence response of E. coli CPS is largely insensitive to ligand binding, changes of the individual probes in intensity, lifetime, anisotropy, and accessibility to acrylamide quenching highlight the dynamic interplay between several protein domains, as well as between subunits. W213 within the carboxy phosphate domain, for example, exhibits an almost 40% increase in intensity upon saturation with ATP; W437 of the oligomerization domain, in contrast, is essentially silent in its fluorescence to the binding of ligands. Nucleotide and bicarbonate association within the large subunit induce fluorescence changes in both W170 and W175 of the small subunit, indicative of the type of long-range interactions purportedly synchronizing the carboxy phosphate and amidotransferase domains of the enzyme to initiate catalysis. ATP and ADP engender different fluorescence responses in most tryptophans, perhaps reflecting coordinating, conformational changes accompanying the cycling of reactants and products during catalysis. † This work was supported by NIH grant P20 RR016478 (to JLJ) from the INBRE Program of the National Center for Research Resources at Southwestern Oklahoma State University, and by NIH grant GM33216 and Welch Foundation grant A1543 (to GDR) at Texas A&M University. Molecular graphics images were produced using the UCSF package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, which is supported by NIH grant P41 RR-01081. *Corresponding authors. Phone: (580) Fax: (580) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptCarbamoyl phosphate synthetase (CPS) 1 from E. coli coordinates five substrates (2 ATP, bicarbonate, glutamine, and water) through a series reactions involving at least three unstable intermediates during the synthesis of its ultimate product, carbamoyl phosphate (1,2). Moreover, crystal structure coordinates have revealed that the reaction mechanism involves three distinct active sites separated by almost 100Å, but connected by a series of intra-and inter-molecular tunnels that presumably shuttle unstable intermediates from site to site (3-5).In the minimal mecha...

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The determination of thermodynamic allosteric parameters of an enzyme undergoing steady-state turnover

Cited by 84 publications

References 10 publications

Kinetic and Structural Characterization of Phosphofructokinase from Lactobacillus bulgaricus

Kinetic and Structural Characterization of Phosphofructokinase from Lactobacillus bulgaricus

Temperature effects on the allosteric responses of native and chimeric aspartate transcarbamoylases 1 1Edited by J. H. Miller

Resolving the Fluorescence Response of Escherichia coli Carbamoyl Phosphate Synthetase: Mapping Intra- and Intersubunit Conformational Changes

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