title: Quaternary structure, Mg2+ interactions, and some kinetic properties of the beta-galactosidase from Thermoanaerobacterium thermosulfurigenes EM1.
abstract: The beta-galactosidase from Thermoanaerobacterium thermosulfurigenes EM1 was found to be a dimer with a monomer molecular weight of about 85,000. It lacks the alpha-peptide and an important alpha-helix that are both needed for dimer-dimer interaction and there is no homology in other important dimer-dimer interaction areas. These differences in structure probably account for the dimeric (rather than tetrameric) structure. Only 0.19 Mg2+ bound per monomer and Mg2+ had only small effects on the activity and heat stability. The absence of residues equivalent to Glu-416 and His-418 (two of the three ligands to Mg2+ in the beta-galactosidase from Escherichia coli) probably accounts for the low level of Mg2+ binding and the consequent lack of response to Mg2+. Both Na+ and K+ also had no effect on the activity. The enzyme activity with o-nitrophenyl-beta-D-galactopyanoside (ONPG) was very similar to that with p-nitrophenyl-beta-D-beta-D-galactopyranoside (PNPG) and the ONPG pH profile was very similar to the PNPG pH profile. These differences are in contrast to the E.coli beta-galactosidase, which dramatically discriminates between these two substrates. The lack of discrimination by the T. thermosulfurigenes beta-galactosidase could be due to the absence of the sequence equivalent to residues 910-1023 of the E. coli beta-galactosidase. Trp-999 is probably of the most importance. Trp-999 of the E. coli beta-galactosidase is important for aglycone binding and ONPG and PNPG differ only in their aglycones. The suggestion that the aglycone site of the T. thermosulfurigenes beta-galactosidase is different was strengthened by competitive inhibition studies. Compared to E. coli beta-galactosidase, D-galactonolactone was a very good inhibitor of the T. thermosulfurigenes enzyme, while L-ribose inhibited poorly. These are transition-state analogs and the results indicate that T. thermosulfurigenes beta-galactosidase binds the transition state differently than does E. coli beta-galactosidase. Methanol and glucose were good acceptors of galactose, and allolactose was formed when glucose was the acceptor. Allolactose could not, however, be detected by TLC when lactose was the substrate. The differences noted may be due to the thermophilic nature of T. thermosulfurigenes.
authors.affiliation: Division of Biochemistry, Faculty of Science, University of Calgary, Canada. huber@acs.ucalgary.ca
authors.forename: R E
authors.initials: RE
authors.lastname: Huber
authors.forename: N J
authors.initials: NJ
authors.lastname: Roth
authors.forename: H
authors.initials: H
authors.lastname: Bahl
mesh_terms: D001421:Bacteria, Anaerobic / Q000201:enzymology*; D001667:Binding, Competitive; D003956:Dialysis; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D005697:Galactosides / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism*; D000432:Methanol / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology; D011487:Protein Conformation; D012964:Sodium / Q000494:pharmacology; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D005697:Galactosides; C001970:allolactose; D012964:Sodium; D001616:beta-Galactosidase; D008274:Magnesium; D005947:Glucose; D007785:Lactose; D011188:Potassium; D000432:Methanol