Contribution of Lys276 to the conformational flexibility of the active site of glutamate decarboxylase from Escherichia coli

Abstract

Glutamate decarboxylase is a pyridoxal 5¢-phosphatedependent enzyme responsible for the irreversible a-decarboxylation of glutamate to yield 4-aminobutyrate. In Escherichia coli, as well as in other pathogenic and nonpathogenic enteric bacteria, this enzyme is a structural component of the glutamate-based acid resistance system responsible for cell survival in extremely acidic conditions (pH < 2.5). The contribution of the active-site lysine residue (Lys276) to the catalytic mechanism of E. coli glutamate decarboxylase has been determined. Mutation of Lys276 into alanine or histidine causes alterations in the conformational properties of the protein, which becomes less flexible and more stable. The purified mutants contain very little (K276A) or no (K276H) cofactor at all. However, apoenzyme preparations can be reconstituted with a full complement of coenzyme, which binds tightly but slowly.The observed spectral changes suggest that the cofactor is present at the active site in its hydrated form. Binding of glutamate, as detected by external aldimine formation, occurs at a very slow rate, 400-fold less than that of the reaction between glutamate and pyridoxal 5¢-phosphate in solution. Both Lys276 mutants are unable to decarboxylate the substrate, thus preventing detailed investigation of the role of this residue on the catalytic mechanism. Several lines of evidence show that mutation of Lys276 makes the protein less flexible and its active site less accessible to substrate and cofactor.

Lysine 238 Is an Essential Residue for α,β-Elimination Catalyzed by Treponema denticola Cystalysin

“…Treatment with NaBH 4 and NaCNBH 3 provides evidence that PLP is bound in these mutants as free aldehyde. Although the absorbing species at 390 -400 nm can be easily attributed to the free aldehyde form of the enzyme-bound PLP, the 320-nm species is probably from the hydrated form of the coenzyme, as previously suggested for active site lysine mutants of D-amino acid transaminase (4), ornithine decarboxylase (8), aminolevulinate synthase (11), and glutamate decarboxylase (25). The emission band at 371 nm following excitation at 320 nm of the reconstituted K238R mutant, together with the finding that, unlike wild-type, K238R does not undergo changes in the absorption spectra of the cofactor depending on pH, is consistent with the attribution of the 320-nm species to a PLP-hydrated form.…”

Mutation of His465 Alters the pH-dependent Spectroscopic Properties of Escherichia coli Glutamate Decarboxylase and Broadens the Range of Its Activity toward More Alkaline pH

“…This behavior contrasts with that of wild type GadB (4,5). The range of analysis was therefore extended to 9.5, and phosphate was used as buffer instead of acetate.…”

“…coli possesses two Gad isoforms, GadA and GadB, which exhibit an acidic pH optimum (pH 3.8 -4.6) and become activated intracellularly because in extremely acidic environments protons leak through the bacterial cell membrane (2). pH-dependent activation of Gad is accompanied by a distinct change in the absorption spectrum of the cofactor (3)(4)(5)(6). At wavelengths above 300 nm, the spectrum changes from one at pH Ͼ 5.3 with the major absorbance band at 340 nm (inactive enzyme) to another at pHϽ 5.3 with the major band at 420 nm (active enzyme).…”

“…The midpoint of the spectroscopic change is shifted from pH 5.3 to 5.8 in the presence of chloride, which is the most abundant anion in gastric secretions (3). The spectral transition exhibits a high level of cooperativity of the protonation/deprotonation process, with at least six protons being involved (3)(4)(5). The 420-nm absorbing species is generally accepted to be the ketoenamine form of the PLP-Lys 276 internal aldimine, protonated on the Schiff base nitrogen (Fig.…”

“…O'Leary and Brummund (7,8) assumed that it is an aldamine in which the C4Ј is substituted by a cysteine residue. Tramonti et al (5), upon mutation of the Lys 276 , proposed that the 340-nm chromophore is the enolimine tautomer of the ketoenamine. Model studies in aqueous and nonpolar solvents have shown that aldimines and aldamines of PLP originate different fluorescence emission spectra when excited at 330 nm (9).…”