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Complementation In Vitro Between Mutationally Altered ß₂ subunits of Escherichia coli Tryptophan Synthetase

Department of Microbiology, Scripps Clinic and Research Foundation, La Jolla, California 92037

ABSTRACT

Cross-reacting ß₂ subunits (CRMs) were purified from eight trpB missense mutants to test for complementation in vitro after urea dissociation and reaggregation. One CRM (B290, demonstrating "repairability," i.e., the appearance of enzymatic activity on combination with subunits) was clearly positive with four others, all "non-repairable" CRMs resulting from mutations at three different but neighboring sites. One complementing pair, B290-B248, was studied in more detail and found, upon mixing purified proteins, to give complementation in the absence of denaturants. Complementation activity was low in each case. To study the mechanism of the modest increases in activity, we used a reduced ß₂ subunit as an artificial CRM to form hybrids where both the amount of activity due to complementation and the amount of hybrid could be measured. (In a reduced ß₂ subunit, the two pyridoxal phosphate cofactors have been chemically reduced by sodium borohydride and are covalently attached to lysine residues. This abolishes activity in the tryptophan synthetic reaction and causes the protein to migrate much faster than normal in acrylamide gel electrophoresis.) Reduced ß₂ subunit formed hybrid dimers with the non-repairable CRMs B244 and B248 at pH 6.0, but no enzymatic activity appeared. On the other hand, when reduced ß₂ subunit was mixed with B290 CRM at pH 6.0 to 6.6, an activity increase was seen that was proportional to the amount of hybrid. We conclude that hybrid formation is essential for complementation and that the mechanism of complementation in this system is the correction of a repairable active site on the B290 ß chain by a conformational change occuring when hybrid dimer is formed. This type of complementation must be restricted to a small class of CRMs having a conformationally deformed active site. From the amount of hybrid present and the increase in activity, a specific activity of 50 U/mg was calculated for the hybrid containing reduced and B290 ß chains. This value is slightly less than but close to the activity of the hybrid formed between reduced and normal ß chains, shown earlier to have half the specific activity of the normal dimer.