SHARED ACTIVE-SITES IN OLIGOMERIC ENZYMES - MODEL STUDIES WITH DEFECTIVE-MUTANTS OF ASPARTATE TRANSCARBAMOYLASE PRODUCED BY SITE-DIRECTED MUTAGENESIS

Many oligomeric enzymes are functional only in the assembled form, and it is often difficult to determine unambiguously why monomers are inactive. In some cases individual monomers cannot fold into stable correct ("native") conformations without contributions from interchain interactions. For other oligomers, catalysis requies the contributions of amino acid residues at the interface between adjacent polypetide chains, and monomers are inactive because they cannot form complete active sites. A test for the presence of shared sites was devised that is based on the formation of active hybrid oligomers from appropriate inactive parenteal mutants produced by site-directed mutagenesis. This approach was applied in a stud of the catalytic trimer of aspartate transcarbamoylase (aspartate carbamoyltransferase, EC 2.1.3.2) from Escherichia coli, using three mutants, in which Ser-52 was replaced by His, Lys-84 by Gln, or His-134 by Ala. Hybrid trimers formed from the virtually inactive Ser and Lys mutants were 105 more active than the parental proteins, and the specific activities of each hybrid were about 33% that of the wild-type trimer, as expected for the scheme based on shared sites. Hybrids from the His and Lys mutants had comparable specific activities. Moreover, one hybrid with .apprxeq. 33% activity had one high-affinity binding site for a bisubstrate analog as compared to about three for wild-type trimer. As a further test, hybrids were also formed from wild-type and double-mutant (Lys-84.fwdarw.Gln and His-134.fwdarw.Ala) trimers. The hybrid containing two chains with the double mutation and one wild-type chain had very little activity, and that composed of one double mutant and two wild-type chains had 32% the specific activity of wild-type trimers. This negative complementation experiment is in quantitative accord with the scheme based on shared sites at or near the interfaces between adjacent chains. The techniques used to demonstrate shared active sites in the catalytic subunits of aspartate transcarbamolyase can be applied generally to various types of oligomers (dimers, tetramers, etc.) to determine whether the participation of amino acid residues from adjoining chains is essential for forming active sites in oligomer enzymes.