CATALYTIC CONSEQUENCES OF EXPERIMENTAL EVOLUTION - CATALYSIS BY A 3RD-GENERATION EVOLVANT OF THE 2ND BETA-GALACTOSIDASE OF ESCHERICHIA-COLI, EBG(ABCDE), AND BY EBG(ABCD) A 2ND-GENERATION EVOLVANT CONTAINING 2 SUPPOSEDLY KINETICALLY SILENT MUTATIONS
S. Krishnan et al., CATALYTIC CONSEQUENCES OF EXPERIMENTAL EVOLUTION - CATALYSIS BY A 3RD-GENERATION EVOLVANT OF THE 2ND BETA-GALACTOSIDASE OF ESCHERICHIA-COLI, EBG(ABCDE), AND BY EBG(ABCD) A 2ND-GENERATION EVOLVANT CONTAINING 2 SUPPOSEDLY KINETICALLY SILENT MUTATIONS, Biochemical journal, 312, 1995, pp. 971-977
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 measure
d. 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, a
s well as two changes hitherto considered to have no kinetic effect, S
er(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)-->L
ys change in the large subunit (e). Comparison of ebg(abcd) With ebg(a
b) [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 fac
tor of 2.5, and also decrease the charge on the aglycone oxygen atom a
t the first transition state; the charge on the glycone, however, is u
naltered [see K, Konstantinidis, Sinnott and Hall (1993) Biochem. J. 2
91, 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] sugg
ests that degalactosylation of a hypothetical ebg(abe) enzyme would be
so slow that the enzyme would have no biological advantage over the a
ncestral ebg(ab). The transfer products from galactosyl-ebg(abcd) and
galactosyl-ebg(abcde) to high concentrations to glucose have been meas
ured; the predominant product is allolactose, but significant quantiti
es of lactose are also formed; however, at apparent kinetic saturation
of the galactosyl-enzyme, hydrolysis rather than transfer is the prep
onderant pathway. A knowledge of the rates of enzyme-catalysed exchang
e 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 eb
g(abcde). As with the other evolvants, changes in the profile away fro
m the rate-determining transition state are essentially random, and th
ere is no correlation between the changes in the free energies of inte
rmediates and of their flanking transition states. We consider the agg
regate of our kinetic data on the ebg system to be telling experimenta
l support for the theoretical objections of Pettersson [Pettersson (19
92) fur. J. Biochem. 206, 289-295 and previous papers] to the Albery-K
nowles theory of the evolution of enzyme kinetic activity.