| Abstract
| - N-Succinyl-ll-diaminopimelateaminotransferase (DAP-AT) (EC 2.6.1.17), a key enzyme in thebacterialpathway to l-lysine, was purified to near homogeneity(1500-fold) in five steps from wild type EscherichiacoliATCC 9637. This pyridoxal phosphate (PLP) dependent enzyme has amolecular weight of 39.9 kDa, appears toform an active homodimer, and uses l-glutamate as the aminogroup donor for its substrate,N-succinyl-α-amino-ε-ketopimelic acid (1a) (Km = 0.18± 0.04 mM, kcat = 86 ± 5s-1). Progress of the reaction ismonitored byspectrophotometric observation of decrease in NADPH concentration at340 nm in a coupled enzyme assay withl-glutamate dehydrogenase (EC 1.4.1.4).Stereochemically pure 1a was synthesized as itstrilithium salt by enereaction of methyl glyoxylate with methylN-succinyl-l-allylglycinate (4a)followed by hydrogenation of the doublebond, Dess−Martin oxidation of the alcohol, and careful lithiumhydroxide hydrolysis. Similar approaches allowedsynthesis of a series of substrate analogues1b−g having differentN-acyl substituents, as well as derivativesmissingthe carboxyl group or the amide functionality (13 and17, respectively). Compounds lacking the ketofunctionality(18a, 18c, and 19) were also prepared.Assay of DAP-AT shows that the enzyme has quite strictrequirements forsubstrate recognition, but it will accept compounds with an aromaticring in place of the terminal succinyl carboxylgroup in 1a (e.g. N-Cbz-α-amino-ε-ketopimelicacid (1c)). Reaction of substrates1a,c with hydrazine hydrate followedby NaCNBH3 reduction gives2-(N-(succinylamino))- (20a) and2-(N-Cbz-amino)-6-hydrazinoheptane-1,7-dioicacids(20c), respectively. These are the most potentslow-binding inhibitors of any DAP-metabolyzing enzymereportedso far (Ki* for DAP-AT: 20a is 22± 4 nM; 20c is 54 ± 9 nM).
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