Serine 948 and threonine 1042 are crucial residues for allosteric regulation of Escherichia coli carbamoylphosphate synthetase and illustrate coupling effects of activation and inhibition pathways

Escherichia coli carbamoylphosphate synthetase (CPSase) is a key enzyme in the pyrimidine nucleotides and arginine biosynthetic pathways. The enzyme h arbors a complex regulation, being activated by ornithine and inosine 5'-mo nophosphate (IMP), and inhibited by UMP. CPSase mutants obtained by in vivo mutagenesis and selected on the basis of particular phenotypes have been c haracterized kinetically. Two residues, serine 948 and threonine 1042, appe ar crucial for allosteric regulation of CPSase. When threonine 1042 is repl aced by an isoleucine residue, the enzyme displays a greatly reduced activa tion by ornithine. The T1042I mutated enzyme is still sensitive to UMP and IMP, although the effects of both regulators are reduced. When serine 948 i s replaced by phenylalanine, the enzyme becomes insensitive to UMP and IMP, but is still activated by ornithine, although to a reduced extent. When co rrelating these observations to the structural data recently reported, it b ecomes clear that both mutations, which are located in spatially distinct r egions corresponding respectively to the ornithine and the UMP/IMP binding sites, have coupled effects on the enzyme regulation. These results provide an illustration that coupling of regulatory pathways occurs within the all osteric subunit of E. coli CPSase. In addition, other mutants have been characterized, which display altered a ffinities for the different CPSase substrates and also slightly modified pr operties towards the allosteric effecters: P165S, P170L, A182V, P360L, S743 N, T800F and G824D. Kinetic properties of these modified enzymes are also p resented here and correlated to the crystal structure of E. coli CPSase and to the phenotype of the mutants. (C) 1999 Academic Press.