Abstract
beta-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 beta-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-beta-galactosidase bound Mg2+ as well as did normal beta-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 beta-galactosidases were less stable to heat than was wild type, but H540N-beta-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.
Highlights
-Galactosidase (-D-galactoside galactohydrolase, EC 3.2.1.23) from Escherichia coli catalyzes hydrolytic and transgalactosylic reactions with -D-galactosides (Huber et al, 1976)
Recent unpublished results1 indicate that His-540 is within H bonding distance of the C6 hydroxyl groups of substrate and transition state analog inhibitors
In this paper we report the importance of the interactions of His-540 of -galactosidase from E. coli with the C6 hydroxyl group of both the ground and the transition states
Summary
-Galactosidase (-D-galactoside galactohydrolase, EC 3.2.1.23) from Escherichia coli catalyzes hydrolytic and transgalactosylic reactions with -D-galactosides (Huber et al, 1976). ␥-D-galactonolactone) or do not have the carbon equivalent to the C1 group that D-galactose has (e.g. L-ribosefuranose form) are good inhibitors of -galactosidase (Lalegerie et al, 1982; Huber and Brockbank, 1987). Their geometries probably resemble the putative planar oxo-carbonium ion transition state. The active sites of enzymes are usually structured to be more complementary to the transition state than to the substrate. In this paper we report the importance (for binding and catalysis) of the interactions of His-540 of -galactosidase from E. coli with the C6 hydroxyl group of both the ground and the transition states
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