The ebg system of E. coli is an ideal vehicle for the study of enzyme evolution, a general problem in evolutionary biology and biochemistry, made pressing in medicine by changing specificities of bacterial antibiotic-detoxifying enzymes. Experiments directed towards the evolution of the ebg system in the laboratory have resulted in the genetic, structural, and biochemical characterization of the functional protein (a beta-galactosidase), and of those biochemical changes consequent upon the limited number of evolutionary changes brought about by spontaneous mutations so far observed. Related experiments involving chemical mutagenesis under the conditions of selection will now address the question as to whether the limited repertoire of amino-acid changes in the small and large subunits of the ebg beta-galactosidase is a consequence of the inherently limited possibilities of the ancestral gene-sequence or a side-effect of the modus operandi of the particular replicative and repair machinery in E. coli. By alteration of the chemical nature of the selecting substrate, selection will be directed towards a particular chemical step or a particular part of the catalytic machinery of the enzyme. The location and type base-changes in the gene will be determined. The free energy profiles of the reactions catalyzed by evolvants will be measured, and the changes compared with the predictions of current theories; particular attention will be paid to the additivity or otherwise of evolutionary changes in the profile. Changes in transition state structure (particularly where selection has been applied to a particular part of the catalytic machinery) will be sought for using established probes of physical organic chemistry.
