Pyruvate Carboxylase: Effect of Reaction Components and Analogues of Acetyl‐Coenzyme A on the Rate of Inactivation in the Presence and Absence of Trinitrobenzene Sulphonate
Authors: SCRUTTON Michael C. , PEARCE P. Helen , FATEBENE Frances 
Published: March 2005

Pyruvate carboxylase purified from chicken liver is inactivated on incubation with amino‐group‐specific reagents, e.g. trinitrobenzene sulphonate. By analogy with previous studies on rat liver pyruvate carboxylase [Scrutton, M. C. and White, M. D. (1973) J. Biol. Chem. 248, 5541–5544], this inactivation which is accompanied by incorporation of approximately 1 trinitrophenyl residue per biotinyl residue is presumed to result from specific loss of acetyl‐CoA‐dependent catalytic activity. The rate of inactivation by trinitrobenzene sulphonate is decreased by addition of acyl derivatives of coenzyme A which either activate or inhibit this pyruvate carboxylase, or of adenine nucleotides. In contrast other substrates and activators e.g. Mg2+ are ineffective. Three observations suggest that protection against inactivation by trinitrobenzene sulphonate results from kinetically significant binding at the allosteric activator site. Agreement is observed between kinetic constants obtained in initial rate and inactivation rate studies. Adenosine 3′,5′‐bisphosphate affords effective protection against inactivation by trinitrobenzene sulphonate. MgATP2−, the true substrate of the pyruvate carboxylase reaction, is less effective in affording such protection than is ATP4−.
No ligand tested affords complete protection against inactivation of chicken liver pyruvate carboxylase by trinitrobenzene sulphonate. The extent of protection observed for the enzyme‐ligand complex is a function of the nature of the ligand, but is independent of the purity or catalytic activity for the enzyme preparation.
The Hill coefficients (nH) characterising the concentration dependence of protection by acyl‐CoA are decreased as compared with those obtained in initial rate studies. This effect is most marked for weaker activators.
Similar results are obtained when inactivation of rat‐liver pyruvate carboxylase is examined in the presence of these ligands, except that the extent of protection observed appears less dependent on the nature of the ligand and, for the more effective activators, nH obtained from the protection data does not differ significantly from that observed in initial rate studies. Acyl derivatives of CoA and CoASH also protect rat‐liver pyruvate carboxylase against inactivation by pyridoxal phosphate with properties similar to those observed for protection against inactivation by trinitrobenzene sulphonate.
We conclude that the lysyl residue which is modified by trinitrobenzene sulphonate is most probably not directly involved in activator binding. The data suggest that this residue is not located in close proximity to the activator site since certain analogues, e.g. acetyl dephospho‐CoA, which act as competitive inhibitors with respect to acetyl‐CoA, fail to afford significant protection against inactivation by trinitrobenzene sulphonate. The properties of protection provide further support for the suggestion that substantial differences exist in the nature of the interactions between the catalytic and activator sites in these two pyruvate carboxylases.
Incubation with millimolar concentrations of adenosine 2′,5′‐bisphosphate and 3′,5′‐bisphosphates causes specific and apparently irreversible inactivation of rat‐liver pyruvate carboxylase. Other adenine nucleotides are less effective. The extent of inactivation is dependent on protein concentration and is decreased by addition either of ATP4‐ (MgATP2−) at a concentration sufficient to saturate the catalytic site, or of high concentrations of sucrose or glycerol. Other reaction components, including acetyl‐CoA, are ineffective. Inactivation induced by adenine nucleotides appears to result from their interaction at a site other than the catalytic or activator site. The mechanism underlying this inactivation does not appear to involve dissociation of the enzyme since the response to addition of reaction components and polyhydroxy compounds differs markedly from that observed for inactivation induced by dilution.