Cooperative binding of the bisubstrate analog N-(phosphonacetyl)-L-aspartate to aspartate transcarbamoylase and the heterotropic effects of ATP and CTP

Journal Article · Tue Feb 14 23:00:00 EST 1989 · J. Biol. Chem.; (United States)

OSTI ID:6470530

Newell, J O; Markby, D W; Schachman, H K

Most investigations of the allosteric properties of the regulatory enzyme aspartate transcarbamoylase (ATCase) from Escherichia coli are based on the sigmoidal dependence of enzyme activity on substrate concentration and the effects of the inhibitor, CTP, and the activator, ATP, on the saturation curves. Interpretations of these effects in terms of molecular models are complicated by the inability to distinguish between changes in substrate binding and catalytic turnover accompanying the allosteric transition. In an effort to eliminate this ambiguity, the binding of the 3H-labeled bisubstrate analog N-(phosphonacetyl)-L-aspartate (PALA) to aspartate transcarbamoylase in the absence and presence of the allosteric effectors ATP and CTP has been measured directly by equilibrium dialysis at pH 7 in phosphate buffer. PALA binds with marked cooperativity to the holoenzyme with an average dissociation constant of 110 nM. ATP and CTP alter both the average affinity of ATCase for PALA and the degree of cooperativity in the binding process in a manner analogous to their effects on the kinetic properties of the enzyme; the average dissociation constant of PALA decreases to 65 nM in the presence of ATP and increases to 266 nM in the presence of CTP while the Hill coefficient, which is 1.95 in the absence of effectors, becomes 1.35 and 2.27 in the presence of ATP and CTP, respectively. The dissociation constant of PALA from the catalytic subunit is 95 nM. Interpretation of these results in terms of a thermodynamic scheme linking PALA binding to the assembly of ATCase from catalytic and regulatory subunits demonstrates that saturation of the enzyme with PALA shifts the equilibrium between holoenzyme and subunits slightly toward dissociation.

• Research Organization: Univ. of California, Berkeley (USA)
• OSTI ID: 6470530
• Journal Information: J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 264:5; ISSN JBCHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
ASPARTIC ACID
ATP
BACTERIA
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL EFFECTS
CARBOXYLIC ACIDS
ENZYME ACTIVITY
ENZYME INHIBITORS
ENZYMES
ESCHERICHIA COLI
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
MICROORGANISMS
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
REACTION KINETICS
TRACER TECHNIQUES
TRANSFERASES
TRITIUM COMPOUNDS