Site-directed fluorescence labeling reveals differences on the R-conformer of glucosamine 6-phosphate deaminase of Escherichia coli induced by active or allosteric site ligands at steady state.
Engineered glucosamine 6-phosphate deaminase of Escherichia coli with unique reactive cysteines at positions 164 or 206 was created by site-directed mutagenesis to monitor the allosteric transition in solution by the fluorescence emission of the bimane or dansyl-amidoethyl groups attached to the indicated residues. The selection of both positions was due to the differential interaction of these residues between T- and R-conformers at the interface of two trimers that form the hexameric structure; in the T-conformer, residue 164 or 206 presents only intrasubunit contacts, but in the R-conformer, new intersubunit contacts are established. As in the wild-type enzyme, fluorescent-labeled mutants show no modification on the allosteric activation of the K-system, only the kcat was reduced to a value of 72 s(-1) (approximately 50% of wild-type). With these preparations, conformational changes were detected by the fluorescence emission spectra at steady state when the active site or the allosteric site ligands were titrated. Despite the similar changes in the fluorescence spectra that were correlated with the induction of the R-state, differences were observed at the maximal change in the fluorescence spectra and in the relative solvent polarities at the positions labeled. These data suggested structural differences in the conformation of the R-state when it is induced from the active site or from the allosteric site, which is not consistent with the two-state structural model proposed by previous crystallographic studies of this enzyme.
Sosa-Peinado
Alejandro
A
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, P.O. Box 70-159, México, DF 04510, México. asosa@bq.unam.mx
González-Andrade
Martín
M
D019747:Aldose-Ketose Isomerases / Q000737:chemistry* / Q000235:genetics; D000495:Allosteric Site / Q000187:drug effects*; D001665:Binding Sites / Q000187:drug effects*; D001643:Bridged Bicyclo Compounds / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D003619:Dansyl Compounds / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D008024:Ligands; D008698:Mesylates / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D001643:Bridged Bicyclo Compounds; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D008024:Ligands; D008698:Mesylates; C508126:dansyl-amidoethyl methanethiosulfonate; C020582:glucosamine-6-phosphate isomerase; D019747:Aldose-Ketose Isomerases; D003545:Cysteine; C028381:monobromobimane