On the functional role of Arg172 in substrate binding and allosteric transition in Escherichia coli glucosamine-6-phosphate deaminase.
Glucosamine-6-phosphate deaminase from Escherichia coli (EC 3.5.99.6) is an allosteric enzyme, activated by N-acetylglucosamine 6-phosphate, which converts glucosamine-6-phosphate into fructose 6-phosphate and ammonia. X-ray crystallographic structural models have showed that Arg172 and Lys208, together with the segment 41-44 of the main chain backbone, are involved in binding the substrate phospho group when the enzyme is in the R activated state. A set of mutants of the enzyme involving the targeted residues were constructed to analyze the role of Arg172 and Lys208 in deaminase allosteric function. The mutant enzymes were characterized by kinetic, chemical, and spectrometric methods, revealing conspicuous changes in their allosteric properties. The study of these mutants indicated that Arg172 which is located in the highly flexible motif 158-187 forming the active site lid has a specific role in binding the substrate to the enzyme in the T state. The possible role of this interaction in the conformational coupling of the active and the allosteric sites is discussed.
Lucumí-Moreno
Armando
A
Laboratorio de Fisicoquímica e Ingeniería de Proteínas, Departamento de Bioquímica, Fac. de Medicina, Universidad Nacional Autónoma de México, Mexico City, DF, Mexico. armando@bq.unam.mx
Calcagno
Mario L
ML
D019747:Aldose-Ketose Isomerases / Q000737:chemistry / Q000378:metabolism*; D000495:Allosteric Site; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001120:Arginine; C020582:glucosamine-6-phosphate isomerase; D019747:Aldose-Ketose Isomerases; D008239:Lysine