title: Structure of glutamate decarboxylase from E. coli: spectral studies.
abstract: Structure of recombinant glutamate decarboxylase (GAD alpha) was studied by optical methods and electron microscopy. The active (pH 4.6) and inert (pH 6.3) holoGAD and apoGAD were investigated. Absorption and CD spectra were recorded in the range of 190 - 500 nm. Visible spectra were resolved into the bands corresponding to individual electron transitions using lognormal curves. The structures of predominant tautomers of internal aldimines were determined as ketoenamine at pH 4.6 and enolimine at pH 6.3. CD spectra show that holoGAD and apoGAD exhibit a negative band at 204 - 245 nm and a positive band near 190 - 204 nm. The contents of the secondary structure elements were estimated on the basis of the values of the mean residue ellipticity. Evidently, the main difference between the GAD forms studied is in the content of alpha-helix and random coil. HoloGAD has 50% of alpha-helix at pH 4.6 and 67% at pH 6.3, whereas apoGAD - 17 and 27%, respectively. Thus presented data establish the essential role of pyridoxal phosphate (PLP) in the organization of the GAD secondary structure due to tightening its polypeptide chain. It seems possible, that conformational changes induced by PLP binding stabilize the protein structure and promote the assembly of subunits into macromolecule, which was confirmed by electron microscopy.
authors.affiliation: V.A. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, 119991 Moscow, Russia.
authors.forename: Natalia P
authors.initials: NP
authors.lastname: Bazhulina
authors.forename: Ekaterina L
authors.initials: EL
authors.lastname: Darii
authors.forename: Vladimir M
authors.initials: VM
authors.lastname: Lobachev
authors.forename: Valery Ya
authors.initials: VY
authors.lastname: Stel'mashchuk
authors.forename: Bella S
authors.initials: BS
authors.lastname: Sukhareva
mesh_terms: D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D005968:Glutamate Decarboxylase / Q000737:chemistry*; D007527:Isoenzymes / Q000737:chemistry; D008854:Microscopy, Electron; D020836:Protein Structure, Quaternary; D013056:Spectrophotometry, Ultraviolet
chemical_list: D007527:Isoenzymes; D005968:Glutamate Decarboxylase