The beta-galactosidase (Escherichia coli) reaction is partly facilitated by interactions of His-540 with the C6 hydroxyl of galactose.
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.
Roth
N J
NJ
Division of Biochemistry, Faculty of Science, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
Huber
R E
RE
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D005690:Galactose / Q000378:metabolism*; D006639:Histidine; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013816:Thermodynamics; D001616:beta-Galactosidase / Q000737:chemistry* / Q000378:metabolism*
D017931:DNA Primers; D006639:Histidine; D001616:beta-Galactosidase; D005690:Galactose