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  • Title: Catalytic consequences of experimental evolution: catalysis by a 'third-generation' evolvant of the second beta-galactosidase of Escherichia coli, ebgabcde, and by ebgabcd, a 'second-generation' evolvant containing two supposedly 'kinetically silent' mutations.
    Author: Krishnan S, Hall BG, Sinnott ML.
    Journal: Biochem J; 1995 Dec 15; 312 ( Pt 3)(Pt 3):971-7. PubMed ID: 8554546.
    Abstract:
    The kinetics of hydrolysis of a series of synthetic substrates by two experimentally evolved forms ('evolvants'), ebgabcd and ebgabcde, of the second beta-galactosidase of Escherichia coli have been measured. The ebgabcd enzyme differs from the wild-type (ebgo) enzyme by Asp92-->Asn (a) and Trp977-->Cys (b) changes in the large subunit, as well as two changes hitherto considered to have no kinetic effect, Ser979-->Gly in the large subunit (c) and Glu122-->Gly in the small subunit (d). The enzyme ebgabcde contains in addition a Glu93-->Lys change in the large subunit (e). Comparison of ebgabcd with ebgab [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 ebgabe enzyme would be so slow that the enzyme would have no biological advantage over the ancestral ebgab. The transfer products from galactosyl-ebgabcd and galactosyl-ebgabcde 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 18O from [1-18O]galactose to water permits the construction of the free-energy profiles for hydrolysis of lactose by begabcd and ebgabcde. 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) Eur. J. Biochem. 206, 289-295 and previous papers] to the Albery-Knowles theory of the evolution of enzyme kinetic activity.
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