The cross-linking by o-phthalaldehyde of two amino acid residues at the active site of 6-phosphogluconate dehydrogenase.
o-phthalaldehyde inactivates homodimeric, NADP+ dependent, 6-phosphogluconate dehydrogenase from sheep liver, upon formation of a single isoindole derivative per enzyme subunit. This indicates that the thiol group of a cysteine residue or the epsilon-amino group of a lysine residue located within 3 A and crosslinked by the reagent is essential for catalysis. Fluorescence analyses of the modified enzyme suggest that the isoindole derivative forms at the binding site of the nicotinamide moiety of NADP+. The enzymes from Trypanosoma brucei and Lactococcus lactis are also inactivated suggesting a similar three-dimensional structure in this domain. The isoindole derivative does not form with two mutants of the T. brucei enzyme (Lys185His and Lys185Leu), this allowing to identify not only the lysine but also the cysteine involved in the cross-linking. The formation of the isoindole derivative inactivates not only the oxidative decarboxylation, but also two partial reactions catalysed by the enzyme.
Giovannini
P P
PP
Department of Biochemistry and Molecular Biology, University, Ferrara, Italy.
Rippa
M
M
Dallocchio
F
F
Tetaud
M
M
Barrett
M P
MP
Hanau
S
S
D000818:Animals; D001665:Binding Sites; D003432:Cross-Linking Reagents; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010734:Phosphogluconate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D012756:Sheep; D013050:Spectrometry, Fluorescence; D014346:Trypanosoma brucei brucei / Q000201:enzymology; D009764:o-Phthalaldehyde / Q000378:metabolism* / Q000494:pharmacology
D003432:Cross-Linking Reagents; D009249:NADP; D009764:o-Phthalaldehyde; D010734:Phosphogluconate Dehydrogenase