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

Abstract

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 alpha-anomer is the true substrate. The imidazole group of this active-site histidine contacts the carboxy groups from Glu148 and Asp141, via its Ndelta1 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.

A Genomic View of Sugar Transport inMycobacterium smegmatisandMycobacterium tuberculosis

“…The first sugar transport cluster in the genome contains genes for a glucosamine isomerase (nagB1), a ␤-glucosidase (bglA), a sugar kinase (sugK), and a complete ABC permease, the latter with distant similarities to other ABC importers. Due to the presence of the metabolic genes, whose products BglA and NagB exhibit 31% and 32% protein identity to homologs of B. subtilis and E. coli, we suggest that the permease catalyzes the uptake of ␤-glucosides such as cellobiose (38,72). It should be noted as well that the closest homologs were the S. coelicolor proteins SCO5236 (NagB homolog with 50% identity) and SCO6670 (BglA homolog with 54% identity).…”

Why Does Escherichia coli Grow More Slowly on Glucosamine than on N -Acetylglucosamine? Effects of Enzyme Levels and Allosteric Activation of GlcN6P Deaminase (NagB) on Growth Rates

“…Some of these mutations alter the catalytic constant of the enzyme and modify qualitatively and quantitatively the allosteric properties of the enzyme, in some cases uncoupling the substrate, GlcN6P, induced cooperativity (homotropic effect), and the activation by the allosteric effector, GlcNAc6P (heterotropic effect). The results obtained have allowed a coherent description of the catalytic mechanism and provide some understanding of the allosteric activation mechanism (1,5,6,8,19,23,24).…”

“…Some of these mutations alter the catalytic constant of the enzyme and modify qualitatively and quantitatively the allosteric properties of the enzyme, in some cases uncoupling the substrate, GlcN6P, induced cooperativity (homotropic effect), and the activation by the allosteric effector, GlcNAc6P (heterotropic effect). The results obtained have allowed a coherent description of the catalytic mechanism and provide some understanding of the allosteric activation mechanism (1,5,6,8,19,23,24).To better understand the functioning of GlcN6P deaminase in vivo and to determine whether the kinetic data obtained with pure enzymes assayed under standardized pH, ionic strength, and temperature conditions in vitro reflect the behavior of the enzyme in the complex milieu of the cell, we studied in vivo several site-directed mutations in nagB. As deaminase is part of an inducible sugar metabolism operon, it was crucial to have the mutations correctly located on the bacterial chromosome so that their expression was regulated in the same way as the wild-type gene.…”

“…The enzymatic mechanism of GlcN6P deamination includes a catalytic triad consisting of Asp141, His143, and Glu148 involved in the sugar ring-opening step, followed by a base-catalyzed enolization reaction involving Asp72 (20,24,26). Mutants with changes at the crucial residues His143 (e.g., His143Gln) and Asp72 (e.g., Asp72Asn) are essentially inactive (their k cat values are only 0.1 and 0.01% of the wild-type values, respectively) (24), and mutations at these positions were not tested in vivo. However, mutants with substitutions that changed the acid residues of Asp141 and Glu148 to their amides were still appreciably active in vitro, although they showed very marked kinetic changes.…”

Allosteric Regulation of Glucosamine-6-Phosphate Deaminase (NagB) and Growth ofEscherichia colion Glucosamine

“…In most organisms, both prokaryotes and eukaryotes, the enzymes are orthologues of the E. coli NagB, with a conserved catalytic triad of readily identifiable amino acids (13). GlcN6P deaminases have been purified from several different sources and found to exist in different quaternary structures.…”