Volume 271, Number 24,
Issue of June 14, 1996
pp. 14296-14301
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
The 
-Galactosidase (Escherichia coli) Reaction Is
Partly Facilitated by Interactions of His-540 with the C6 Hydroxyl of
Galactose
(Received for publication, February 7, 1996)
Nathan J.
Roth
and
Reuben E.
Huber
From the Division of Biochemistry, Faculty of Science, University
of Calgary, Calgary, Alberta T2N 1N4, Canada
-Galactosidases with substitutions for His-540
were only poorly reactive with galactosyl substrates. However, the
activity with substrates that were like galactose but did not have a C6
hydroxyl group was not decreased much as a result of such
substitutions. The loss of transition state stabilization for
galactosyl substrates as a result of substitution was between 
15.4
and 22.8 kJ/mol but only between +0.34 and 6.5 for substrates that
were identical to galactose but lacked the C6 hydroxyl. These findings
indicate that an important function of His-540 is to aid in the
stabilization of the transition state by forming a stable interaction
with the C6 hydroxyl group. This suggestion was strengthened by the
results of competitive inhibition studies showing that
L-arabinolactone (a transition state analog inhibitor of
-galactosidase without a C6 hydroxymethyl group) was bound as well
by the substituted enzymes as by wild type, whereas transition state
analog inhibitors that contain C6 hydroxyls (L-ribose and
D-galactonolactone) were bound much more poorly by the
substituted enzymes than by the wild type enzyme. Substrate analog
inhibitor studies showed that His-540 was also important for binding
interactions with the C6 hydroxyl group of the ground (substrate)
state. The activation by Mg2+ was the same for the
substituted enzymes as for the wild type, and equilibrium dialysis
showed that H540F--galactosidase bound Mg2+ as well as
did normal -galactosidase. The k2 and
Ks values seem to have the same pH interactions as
wild type enzyme, whereas the k3 interactions
are affected differently by pH in the substituted enzymes than in the
wild type enzyme. The rate of the ``degalactosylation'' reaction was
affected more by substitutions for His-540 than was the rate of the
``galactosylation'' reaction. All three substituted
-galactosidases were less stable to heat than was wild type, but
H540N--galactosidase was somewhat more stable than the other two
substituted enzymes. There were some differences in activity and
inhibitory properties that resulted from the different
substitutions.
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