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The cross‐linking by o‐phthalaldehyde of two amino acid residues at the active site of 6‐phosphogluconate dehydrogenase
Author(s) -
Giovannini P. Paolo,
Rippa Mario,
Dallocchio Franco,
Tetaud Manuel,
Barrett Michael P.,
Hanau Stefania
Publication year - 1997
Publication title -
iubmb life
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.132
H-Index - 113
eISSN - 1521-6551
pISSN - 1521-6543
DOI - 10.1080/15216549700203921
Subject(s) - isoindole , chemistry , enzyme , biochemistry , cysteine , dehydrogenase , stereochemistry , oxidoreductase , lysine , residue (chemistry) , dtnb , active site , moiety , amino acid , glutathione
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 ε‐amino group of a lysine residue located within 3 Å and crosslinked by the reagent is essenthial 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.