title: Asymmetric allosteric activation of Escherichia coli glucosamine-6-phosphate deaminase produced by replacements of Tyr 121.
abstract: Tyrosine 121, a residue located in a alpha-helical polypeptide segment of glucosamine 6-phosphate deaminase from Escherichia coli, has recently been proposed to have a role in the binding of the allosteric activator N-acetyl-D-glucosamine 6-phosphate. Accordingly, the site-directed mutants Tyr 121-Thr and Tyr 121-Trp were constructed, to assess experimentally the role of Tyr 121 in the allosteric function of the enzyme. The kinetic study of both mutant forms revealed that the replacements caused striking changes in allosteric activator binding and allosteric properties, when compared to the wild-type enzyme. While the wild-type deaminase behaves as a classical allosteric K-system which can be described by the allosteric concerted model, both mutant forms present an asymmetric behavior toward the allosteric activator, which can be described as two distinct half-of-the-sites allosteric activation steps occurring with different affinities for the N-acetyl-D-glucosamine 6-phosphate. During the first (high affinity) activation phase, the mutant forms of deaminase behave as mixed K/V allosteric enzyme. The biphasic activation curve was also demonstrated by direct binding measurements of the 14C-labeled activator to Tyr 121-Trp and Tyr 121-Thr deaminases. The kinetic analysis of these mutant forms also showed that the threonine replacement produced an important distortion of the enzyme structure reflected in a considerable decrease of its catalytic efficiency.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City, D.F.
authors.forename: M M
authors.initials: MM
authors.lastname: Altamirano
authors.forename: J A
authors.initials: JA
authors.lastname: Plumbridge
authors.forename: E
authors.initials: E
authors.lastname: Horjales
authors.forename: M L
authors.initials: ML
authors.lastname: Calcagno
mesh_terms: D019747:Aldose-Ketose Isomerases; D000494:Allosteric Regulation; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D007527:Isoenzymes; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
chemical_list: D007527:Isoenzymes; D014443:Tyrosine; C020582:glucosamine-6-phosphate isomerase; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases