title: The contribution of a conformationally mobile, active site loop to the reaction catalyzed by glutamate semialdehyde aminomutase.
abstract: The behavior of glutamate semialdehyde aminomutase, the enzyme that produces 4-aminolevulinate for tetrapyrrole synthesis in plants and bacteria, is markedly affected by the extent to which the central intermediate in the reaction, 4,5-diaminovalerate, is allowed to dissociate. The kinetic properties of the wild-type enzyme are compared with those of a mutant form in which a flexible loop, that reversibly plugs the entrance to the active site, has been deleted by site-directed mutagenesis. The deletion has three effects. The dissociation constant for diaminovalerate is increased approximately 100-fold. The catalytic efficiency of the enzyme, measured as k(cat)/K(m) in the presence of saturating concentrations of diaminovalerate, is lowered 30-fold to 2.1 mM(-1) s(-1). During the course of the reaction, which begins with the enzyme in its pyridoxamine form, the mutant enzyme undergoes absorbance changes not seen with the wild-type enzyme under the same conditions. These are proposed to be due to abortive complex formation between the pyridoxal form of the enzyme (formed by dissociation of diaminovalerate) and glutamate semialdehyde itself.
authors.affiliation: Cardiff School of Biosciences, University of Wales, Cardiff CF1 3US, United Kingdom.
authors.forename: R
authors.initials: R
authors.lastname: Contestabile
authors.forename: S
authors.initials: S
authors.lastname: Angelaccio
authors.forename: R
authors.initials: R
authors.lastname: Maytum
authors.forename: F
authors.initials: F
authors.lastname: Bossa
authors.forename: R A
authors.initials: RA
authors.lastname: John
mesh_terms: D000599:Amino Acids, Diamino / Q000737:chemistry; D001665:Binding Sites; D002942:Circular Dichroism; D000458:Cyanobacteria; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D013058:Mass Spectrometry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017384:Sequence Deletion; D013053:Spectrophotometry
chemical_list: D000599:Amino Acids, Diamino; C074874:4,5-diaminopentanoic acid; D019751:Intramolecular Transferases; C061049:glutamate-1-semialdehyde 2,1-aminomutase