Formation of Active Aspartate Transcarbamoylase from Defective Polypeptide Chains Produced by Site-Directed Mutagenesis

Although the genetics literature is replete with exquisite examples of interallelic complementation (1–3) in bacterial strains containing two different mutant copies of the same gene, there is little precise structural information illustrating how functional enzyme complexes are formed by the noncovalent interaction between defective polypeptide chains. For a protein chemist or an enzymologist, interpretation of the results of complementation experiments in terms of molecular mechanisms requires both the availability of suitable mutants containing known defects as well as knowledge of the tertiary and quaternary structures of the wild-type enzyme. Indeed, as Fincham (1) stated in 1966 in the preface to his book: “The present consensus may be summarized by saying that allelic complementation is basically irrelevant to primary gene action, except insofar as it confuses the investigator, but that it is of considerable importance in providing an insight into the structure and functions of multimeric protein molecules.” Because so much is known about the regulatory enzyme aspartate transcarbamoylase (ATCase, aspartate carbamoyltransferase, carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) from Escherichia coli, it can serve as a useful model system for mechanistic studies of interallelic complementation. A large variety of complementing mutants are available (4), and the three-dimensional structure of the wild-type enzyme is known from x-ray crystallography (5, 6). It seemed likely therefore that in vivo and in vitro complementation experiments with various mutations in pyrB, which encodes the catalytic chains of ATCase, would provide valuable information about the folding of the chains into domains, help to identify amino acid residues critical for catalysis, and contribute to our understanding of the nature of the active sites in the enzyme.