title: Evidence for two different mechanisms triggering the change in quaternary structure of the allosteric enzyme, glucosamine-6-phosphate deaminase.
abstract: The generation and propagation of conformational changes associated with ligand binding in the allosteric enzyme glucosamine-6-phosphate deaminase (GlcN6P deaminase, EC 3.5.99.6) from Escherichia coli were analyzed by fluorescence measurements. Single-tryptophan mutant forms of the enzyme were constructed on the basis of previous structural and functional evidence and used as structural-change probes. The reporter residues were placed in the active-site lid (position 174) and in the allosteric site (254 and 234); in addition, signals from the natural Trp residues (15 and 224) were also studied as structural probes. The structural changes produced by the occupation of either the allosteric or the active site by site-specific ligands were monitored through changes in the spectral center of mass (SCM) of their steady-state emission fluorescence spectra. Binding of the allosteric activator produces only minimal signals in titration experiments. In contrast, measurable spectral signals were found when the active site was occupied by a dead-end inhibitor. The results reveal that the two binary complexes, enzyme-activator (R(A)) and enzyme-inhibitor (R(S)) complexes, have structural differences and that they also differ from the ternary complex (R(AS)). The mobility of the active-site lid motif is shown to be independent of the allosteric transition. The active-site ligand induces cooperative SCM changes even in the enzyme-activator complex, indicating that the propagation pathway of the conformational relaxation triggered from the active site is different from that involved in the heterotropic activation. Analysis of the complete set of mutants shows that the occupation of the active site generates structural perturbations, which are propagated to the whole of the monomer and extend to the other subunits. The accumulative effect of these propagated changes should be responsible for the change in the sign of the DeltaG degrees ' of the T to R transition associated with the progression of the active-site occupation, resulting in the predominance of the R over the T forms in the population of deaminase hexamers.
authors.affiliation: Laboratorio de Fisicoquímica e Ingeniería de Proteínas, Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad Universitaria, Mexico City 04510, Mexico. ismaelb@ servidor.unam.mx
authors.forename: Ismael
authors.initials: I
authors.lastname: Bustos-Jaimes
authors.forename: Montserrat
authors.initials: M
authors.lastname: Ramírez-Costa
authors.forename: Lorena
authors.initials: L
authors.lastname: De Anda-Aguilar
authors.forename: Pilar
authors.initials: P
authors.lastname: Hinojosa-Ocaña
authors.forename: Mario L
authors.initials: ML
authors.lastname: Calcagno
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000737:chemistry; D019747:Aldose-Ketose Isomerases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D000494:Allosteric Regulation / Q000235:genetics; D000495:Allosteric Site / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary / Q000235:genetics; D013012:Sorbitol / Q000031:analogs & derivatives* / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity / Q000235:genetics; D013403:Sugar Phosphates / Q000737:chemistry; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics
chemical_list: C099009:2-deoxy-2-aminoglucitol-6-phosphate; D029968:Escherichia coli Proteins; D008024:Ligands; D013403:Sugar Phosphates; C010004:N-acetylglucosamine 6-phosphate; D013012:Sorbitol; D014364:Tryptophan; C020582:glucosamine-6-phosphate isomerase; D019747:Aldose-Ketose Isomerases; D000117:Acetylglucosamine