On the role of the conformational flexibility of the active-site lid on the allosteric kinetics of glucosamine-6-phosphate deaminase.
The active site of glucosamine-6-phosphate deaminase from Escherichia coli (GlcN6P deaminase, EC 3.5.99.6) has a complex lid formed by two antiparallel beta-strands connected by a helix-loop segment (158-187). This motif contains Arg172, which is a residue involved in binding the substrate in the active-site, and three residues that are part of the allosteric site, Arg158, Lys160 and Thr161. This dual binding role of the motif forming the lid suggests that it plays a key role in the functional coupling between active and allosteric sites. Previous crystallographic work showed that the temperature coefficients of the active-site lid are very large when the enzyme is in its T allosteric state. These coefficients decrease in the R state, thus suggesting that this motif changes its conformational flexibility as a consequence of the allosteric transition. In order to explore the possible connection between the conformational flexibility of the lid and the function of the deaminase, we constructed the site-directed mutant Phe174-Ala. Phe174 is located at the C-end of the lid helix and its side-chain establishes hydrophobic interactions with the remainder of the enzyme. The crystallographic structure of the T state of Phe174-Ala deaminase, determined at 2.02 A resolution, shows no density for the segment 162-181, which is part of the active-site lid (PDB 1JT9). This mutant form of the enzyme is essentially inactive in the absence of the allosteric activator, N-acetylglucosamine-6-P although it recovers its activity up to the wild-type level in the presence of this ligand. Spectrometric and binding studies show that inactivity is due to the inability of the active-site to bind ligands when the allosteric site is empty. These data indicate that the conformational flexibility of the active-site lid critically alters the binding properties of the active site, and that the occupation of the allosteric site restores the lid conformational flexibility to a functional state.
Bustos-Jaimes
Ismael
I
Laboratorio de Fisicoquímica e Ingeniería de Proteínas, Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, P.O. Box 70-159, Ciudad Universitaria, Mexico City, D.F. 04510, Mexico. ismaelb@servidor.unam.mx
Sosa-Peinado
Alejandro
A
Rudiño-Piñera
Enrique
E
Horjales
Eduardo
E
Calcagno
Mario L
ML
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D019747:Aldose-Ketose Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000494:Allosteric Regulation; D000495:Allosteric Site; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D018583:Pliability; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D010649:Phenylalanine; C020582:glucosamine-6-phosphate isomerase; D019747:Aldose-Ketose Isomerases; D000409:Alanine