Allosteric Signal Transmission Involves Synergy between Discrete Structural Units of the Regulatory Subunit of Aspartate Transcarbamoylase

Abstract

Previous studies have shown that the S5′ β-strand (r93-r97) of the regulatory polypeptides of the aspartate transcarbamoylases (ATCases) from Serratia marcescens and Escherichia coli are responsible for their diverged allosteric regulatory patterns, including conversion of CTP from an inhibitor in E. coli to an activator in S. marcescens. Similarly, mutation of residues located in the interface between the allosteric and the zinc domains resulted in conversion of the ATP responses of the E. coli enzyme from activation to inhibition, suggesting that this interface not only mediates but also discriminates the allosteric responses of ATP and CTP. To further decipher the roles and the interrelationships of these regions in allosteric communication, allosteric-zinc interface mutations (Y77F and V106A) have been introduced into both the native and the S5′ β-strand chimeric backgrounds. While the significance of this interface in the allosteric regulation has been confirmed, there is no direct evidence supporting the presence of distinct pathways for the ATP and CTP signals through this interface. The analysis of the mutational effects reported here suggested that the S5′ β-strand transmits the allosteric signal by modulating the hydrophobic allosteric-zinc interface rather than disturbing the allosteric ligand binding. Intragenic suppression by substitutions in the hydrophobic interface between the allosteric and the zinc domains of the regulatory chains resulted in the partial recovery of allosteric responses in the EC:rS5'sm chimera and reduced the activation by ATP in the Sm:rS5'ec chimera. Thus, it seems that there is a synergy between these two Structural Units. KeywordsATCase; allosterism; S5′ β-strand; allosteric-zinc interface; mutational effect; signal transmission; ligand binding Aspartate transcarbamoylase (EC 2.1.3.2; ATCase) 2 catalyzes the condensation of Laspartate with carbamoyl phosphate to form carbamoylaspartate and phosphate, ultimately leading to the pyrimidine end products, UTP and CTP (1-3). In Escherichia coli, the holoenzyme is composed of two catalytic subunits and three regulatory subunits (2c 3 :3r 2 ). Three catalytic polypeptides associate to form a functional catalytic subunit (trimer, c 3 ) and two regulatory polypeptides form a regulatory subunit (dimer, r 2 ) (Fig. 1) polypeptide is folded into an allosteric effector-binding domain (Allo domain, r1-r100) and a zinc-binding domain (Zn domain, r105-r146). The active sites are located at the interfaces of the Asp and CP domains of the catalytic trimers, positioned 60 Å away from the nucleotide-binding sites, which are located adjacent to the five-stranded β-sheets of the Allo domains and directly linked to the Zn domains through the S5′ β-strand. The hydrophobic allosteric-zinc interface includes leucine r32, phenylalanine r33, tyrosine r77, and valine r106. These residues are invariant among the four members of the Enterobacteriaceae whose sequences have been reported: E. coli, Serratia marcescens, Erwinia herbicola, and Salmone...

Allosteric Regulation of Catalytic Activity: Escherichia coli Aspartate Transcarbamoylase versus Yeast Chorismate Mutase

“…For both enzymes, substitution of some specific single amino acid residues can be sufficient to change the intensity of allosteric regulation. However, other authors found that these substitutions need not necessarily be positioned at a defined path for signal transduction but might cause global conformational changes due to changes in free energy (3,63).…”

“…The nucleotides might change the stability of the interfaces as part of the primary effect, thereby influencing T-R transition on aspartate binding. On the basis of this, Liu et al proposed that changes in interfaces could influence allostery by altering the global energy of ATCase (63). ATP might induce a signal transduction chain via the R1-C1 interface, while CTP exerts its function via the R1-C4 interface (111).…”