CATALYTIC CONSEQUENCES OF EXPERIMENTAL EVOLUTION - CATALYSIS BY A THIRD-GENERATION EVOLVANT OF THE SECOND BETA-GALACTOSIDASE OF ESCHERICHIA COLI, EBG(ABCDE), AND BY EBG(ABCD) A SECOND-GENERATION EVOLVANT CONTAINING TWO SUPPOSEDLY KINETICALLY SILENT M

摘要

The kinetics of hydrolysis of a series of synthetic substrates by two experimentally evolved forms ('evolvants'), ebg(abcd) and ebg(abcde), of the second beta-galactosidase of Escherichia coli have been measured. The ebg(abcd) enzyme differs from the wild-type (ebg(o)) enzyme by Asp92-->Asn (a) and Trp(977)-->Cys (b) changes in the large subunit, as well as two changes hitherto considered to have no kinetic effect, Ser(979)-->Gly in the large subunit (c) and Glu(122)-->Gly in the small subunit (d). The enzyme ebg(abcde) contains in addition a Glu(93)-->Lys change in the large subunit (e). Comparison of ebg(abcd) With ebg(ab) [Elliott, K, Sinnott, Smith, Bommuswamy, Guo, Hall and Zhang (1992) Biochem. J. 282, 155-164] indicates that the c and d changes in fact accelerate the hydrolysis of the glycosyl-enzyme intermediate by a factor of 2.5, and also decrease the charge on the aglycone oxygen atom at the first transition state; the charge on the glycone, however, is unaltered [see K, Konstantinidis, Sinnott and Hall (1993) Biochem. J. 291, 15-17]. The e mutation causes a fall in the degalactosylation rate of about a factor of 3, and its occurrence only together with c and d mutations [Hall, Betts and Wootton (1989) Genetics 123, 635-648] suggests that degalactosylation of a hypothetical ebg(abe) enzyme would be so slow that the enzyme would have no biological advantage over the ancestral ebg(ab). The transfer products from galactosyl-ebg(abcd) and galactosyl-ebg(abcde) to high concentrations to glucose have been measured; the predominant product is allolactose, but significant quantities of lactose are also formed; however, at apparent kinetic saturation of the galactosyl-enzyme, hydrolysis rather than transfer is the preponderant pathway. A knowledge of the rates of enzyme-catalysed exchange of O-18 from [1-O-18]galactose to water permits the construction of the free-energy profiles for hydrolysis of lactose by beg(abcd) and ebg(abcde). As with the other evolvants, changes in the profile away from the rate-determining transition state are essentially random, and there is no correlation between the changes in the free energies of intermediates and of their flanking transition states. We consider the aggregate of our kinetic data on the ebg system to be telling experimental support for the theoretical objections of Pettersson [Pettersson (1992) fur. J. Biochem. 206, 289-295 and previous papers] to the Albery-Knowles theory of the evolution of enzyme kinetic activity.