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Volume 271, Number 24, Issue of June 14, 1996 pp. 14296-14301
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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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)

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 K_s 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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