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The ABRF-MIRG'02 study: assembly state, thermodynamic, and kinetic analysis of an enzyme/inhibitor interaction.

ABSTRACT: Fully characterizing the interactions involving biomolecules requires information on the assembly state, affinity, kinetics, and thermodynamics associated with complex formation. The analytical technologies often used to measure biomolecular interactions include analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). In order to evaluate the capabilities of core facilities to implement these technologies, the Association of Biomolecular Resource Facilities (ABRF) Molecular Interactions Research Group (MIRG) developed a standardized model system and distributed it to a panel of AUC, ITC, and SPR operators. The model system was composed of a well-characterized enzyme-inhibitor pair, namely bovine carbonic anhydrase II (CA II) and 4-carboxybenzenesulfonamide (CBS). Study participants were asked to measure one or more of the following: (1) the molecular mass, homogeneity, and assembly state of CA II by AUC; (2) the affinity and thermodynamics for complex formation by ITC; and (3) the affinity and kinetics of complex formation by SPR. The results from this study provide a benchmark for comparing the capabilities of individual laboratories and for defining the utility of the different instrumentation.

SUBMITTER: Myszka DG

PROVIDER: S-EPMC2279960 | biostudies-literature | 2003 Dec

REPOSITORIES: biostudies-literature

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The ABRF-MIRG'02 study: assembly state, thermodynamic, and kinetic analysis of an enzyme/inhibitor interaction.

Myszka D G DG Abdiche Y N YN Arisaka F F Byron O O Eisenstein E E Hensley P P Thomson J A JA Lombardo C R CR Schwarz F F Stafford W W Doyle M L ML

Journal of biomolecular techniques : JBT 20031201 4

Fully characterizing the interactions involving biomolecules requires information on the assembly state, affinity, kinetics, and thermodynamics associated with complex formation. The analytical technologies often used to measure biomolecular interactions include analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). In order to evaluate the capabilities of core facilities to implement these technologies, the Association of Biomolecular ...[more]

PMID: 14715884

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Project description:1. The naturally occurring ATPase (adenosine triphosphatase)-inhibitor protein, from bovine heart mitochondria, was obtained as a single pure protein. It was not identical with any of the five subunits (alpha-epsilon) of the isolated ATPase, and appeared to be a single polypeptide chain. 2. The inhibitor combined with the ATPase in a 1:1 molar ratio, producing a completely inhibited ATPase molecule. The affinity of the ATPase for its inhibitor is high; the K(d) is of the order of 10(-8)m. 3. The enthalpy of the ATPase-inhibitor complex-formation is positive, the value of K(d) decreasing as the temperature is raised. This suggests that the forces involved are largely hydrophobic in nature. 4. Hydrolysis of a nucleoside triphosphate promoted formation of the ATPase-inhibitor complex, although the equilibrium position was almost unaffected by the rate of hydrolysis. At low salt concentration, less than 200 turnovers of the ATPase suffice for the ATPase to combine with the inhibitor protein. At higher salt concentrations, a larger number of turnovers is required. It is suggested that the inhibitor binds to a form of the ATPase that is produced transiently during hydrolysis. 5. In the presence of 75mm-K(2)SO(4), the rates of association and dissociation are slow enough to allow their kinetics to be studied. Association is first-order in inhibitor concentration, but fractional order in ATPase concentration. Dissociation is first-order in ATPase-inhibitor complex concentration. The temperature coefficients of the ;on' and ;off' processes were also measured. 6. A simple kinetic model for the ATPase-inhibitor interaction is proposed that can be extended to take into account release of inhibitor protein under energized conditions on the membrane. 7. The isolated ATPase is inhibited by preincubation with Mg(2+), reversible by subsequent addition of EDTA, and by ADP, reversible by subsequent addition of ATP. These effects are not found on the membrane-bound ATPase. The mechanism of these effects is discussed.

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The ABRF-MIRG'02 study: assembly state, thermodynamic, and kinetic analysis of an enzyme/inhibitor interaction.

Publications

The ABRF-MIRG'02 study: assembly state, thermodynamic, and kinetic analysis of an enzyme/inhibitor interaction.

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