title: Affinity labeling of the Escherichia coli aspartate-beta-semialdehyde dehydrogenase with an alkylating coenzyme analogue. Half-site reactivity and competition with the substrate alkylating analogue.
abstract: 3-Chloroacetylpyridine-adenine dinucleotide phosphate (clac3PdADP+, a NADP+ alkylating analogue, irreversibly inactivates aspartate-beta-semialdehyde dehydrogenase with pseudo-first-order kinetics. NADP+ and NADPH, but not the substrate, protected the enzyme from inactivation. The pH dependence of the inactivation kinetics was determined. Incorporation of 1 mol cl[14C]-ac3PdADP+/dimer totally inactivates the enzyme. Successive alkylation by the coenzyme analogue and by the substrate analogue, L-2-amino-4-oxo-5-chloropentanoic acid, was studied. After inactivation with the coenzyme analogue, no incorporation of the substrate analogue was detected. However, when the enzyme was first inactivated with the substrate analogue, the protein could subsequently be alkylated with the coenzyme analogue. The binding of NADP+ and NADPH to aspartate-beta-semialdehyde dehydrogenase was determined by fluorescence.
mesh_terms: D000345:Affinity Labels; D001223:Aspartate-Semialdehyde Dehydrogenase / Q000378:metabolism*; D001665:Binding Sites; D003085:Colic / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009249:NADP / Q000031:analogs & derivatives*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
chemical_list: C017839:3-chloroacetylpyridine adenine dinucleotide phosphate; D000345:Affinity Labels; D009249:NADP; D001223:Aspartate-Semialdehyde Dehydrogenase