CROSS-LINKING BY O-PHTHALALDEHYDE OF 2 AMINO-ACID-RESIDUES AT THE ACTIVE-SITE OF 6-PHOSPHOGLUCONATE DEHYDROGENASE

o-phthalaldehyde inactivates homodimeric, NADP(+) dependent, 6-phospho gluconate dehydrogenase from sheep liver, upon formation of a single i soindole derivative per enzyme subunit. This indicates that the thiol group of a cysteine residue or the epsilon-amino group of a lysine res idue located within 3 Angstrom and crosslinked by the reagent is essen thial for catalysis. Fluorescence analyses of the modified enzyme sugg est that the isoindole derivative forms at the binding site of the nic otinamide moiety of NADP(+). The enzymes from Trypanosoma brucei and L actococcus lactis are also inactivated suggesting a similar three-dime nsional structure in this domain. The isoindole derivative does not fo rm with two mutants of the T. brucei enzyme (Lys185His and Lys185Leu), this allowing to identify not only the lysine but also the cysteine i nvolved in the cross-linking. The formation of the isoindole derivativ e inactivates not only the oxidative decarboxylation, but also two par tial reactions catalysed by the enzyme.