On the Multiple Functional Roles of the Active Site Histidine in Catalysis and Allosteric Regulation of Escherichia coli Glucosamine 6-Phosphate Deaminase

• Published in: Biochemistry (Easton) 2001-08, Vol.40 (34), p.10187-10196
• Main Authors: Montero-Morán, Gabriela M, Lara-González, Samuel, Álvarez-Añorve, Laura I, Plumbridge, Jacqueline A, Calcagno, Mario L
• Format: Article
• Language: English
• Subjects: Aldose-Ketose Isomerases - chemistry; Aldose-Ketose Isomerases - metabolism; Allosteric Regulation; Amino Acid Sequence; Amino Acid Substitution; Animals; Bacteria - enzymology; Binding Sites; Caenorhabditis elegans - enzymology; Catalysis; Cricetinae; Crystallography, X-Ray; Drosophila melanogaster - enzymology; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - metabolism; Escherichia coli - enzymology; Histidine; Humans; Kinetics; Mesocricetus; Mice; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Structure, Secondary; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Sequence Alignment; Sequence Homology, Amino Acid; Sorbitol - analogs & derivatives; Sorbitol - chemistry; Sorbitol - metabolism; Sugar Phosphates - chemistry; Sugar Phosphates - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0105835

The active site of glucosamine-6-phosphate deaminase (EC 3.5.99.6, formerly 5.3.1.10) from Escherichia coli was first characterized on the basis of the crystallographic structure of the enzyme bound to the competitive inhibitor 2-amino-2-deoxy-glucitol 6-phosphate. The structure corresponds to the R allosteric state of the enzyme; it shows the side-chain of His143 in close proximity to the O5 atom of the inhibitor. This arrangement suggests that His143 could have a role in the catalysis of the ring-opening step of glucosamine 6-phosphate whose α-anomer is the true substrate. The imidazole group of this active-site histidine contacts the carboxy groups from Glu148 and Asp141, via its Nδ1 atom [Oliva et al. (1995) Structure 3, 1323−1332]. These interactions change in the T state because the side chain of Glu148 moves toward the allosteric site, leaving at the active site the dyad Asp141-His143 [Horjales et al. (1999) Structure 7, 527−536]. In this research, a dual approach using site-directed mutagenesis and controlled chemical modification of histidine residues has been used to investigate the role of the active-site histidine. Our results support a multifunctional role of His143; in the forward reaction, it is involved in the catalysis of the ring-opening step of the substrate, glucosamine 6-P. In the reverse reaction, the substrate fructose 6-P binds in its open chain, carbonylic form. The role of His143 in the binding of both glucosamine 6-P and reaction intermediates in their extended-chain forms was demonstrated by binding experiments using the reaction intermediate analogue, 2-amino-2-deoxy-d-glucitol 6-phosphate. His143 was also shown to be a critical residue for the conformational coupling between active and allosteric sites. From the pH dependence of the reactivity of the active site histidine to diethyl dicarbonate, we observed a pK a change of 1.2 units to the acid side when the enzyme undergoes the allosteric T to R transition during which the side chain of Glu148 moves toward the active site. The kinetic study of the Glu148-Gln mutant deaminase shows that the loss of the carboxy group and its replacement with the corresponding amide modifies the k cat versus pH profile of the enzyme, suggesting that the catalytic step requiring the participation of His143 has become rate-limiting. This, in turn, indicates that the interaction Glu148-His143 in the wild-type enzyme in the R state contributes to make the enzyme functional over a wide pH range.