title: Beta-galactosidases (Escherichia coli) with double substitutions show that Tyr-503 acts independently of Glu-461 but cooperatively with Glu-537.
abstract: Beta-galactosidases with single substitutions for Tyr-503, Glu-461, and Glu-537 and with double substitutions for Tyr-503 and either Glu-461 or Glu-537 were constructed. Control experiments showed that the very low kcat values obtained for the double-substituted enzymes were not a result of contamination, reversion, or nonactive site activity catalyzed on the surface of the proteins. Circular dichroism studies showed that the structures of the enzymes were intact. E461Q/Y503F-beta-galactosidase was inactivated in an "additive" manner. This indicated that Glu-461 and Tyr-503 act independently in catalysis. Because these residues are at opposite sides of the active site and act in different steps, this is expected. E537D/Y503F-beta-galactosidase was only inactivated a few-fold more than the most inactive of its two single-substituted constituent beta-galactosidases. This showed that Glu-537 and Tyr-503 interact cooperatively on the same step. This correlates well with the proposed role of Tyr-503 as an acid catalyst for the breakage of the covalent bond between Glu-537 and galactose.
authors.affiliation: Division of Biochemistry, Faculty of Science, University of Calgary, Calgary, Alberta, Canada T2N 1N4.
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Roth
authors.forename: Robert M
authors.initials: RM
authors.lastname: Penner
authors.forename: Reuben E
authors.initials: RE
authors.lastname: Huber
mesh_terms: D001665:Binding Sites; D002942:Circular Dichroism; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005690:Galactose / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry*; D005990:Glycerol; D006358:Hot Temperature; D000432:Methanol; D016297:Mutagenesis, Site-Directed; D014443:Tyrosine / Q000737:chemistry*; D001616:beta-Galactosidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D018698:Glutamic Acid; D014443:Tyrosine; D001616:beta-Galactosidase; D005990:Glycerol; D005690:Galactose; D000432:Methanol