N-Succinyl-LL-diaminopimelate aminotransferase (DAP-AT) (EC 2.6.1.17), a key enzyme in the bacterial pathway to L-lysine, was purified to near homogeneity (1500-fold) in five steps from wild type Escherichia coil ATCC 9637. This pyridoxal phosphate (PLP) dependent enzyme has a molecular weight of 39.9 kDa, appears to form an active homodimer, and uses L-glutamate as the amino group donor for its substrate, N-succinyl-alpha-amino-epsilon-ketopimelic acid (1a) (K-m = 0.18 +/- 0.04 mM, k(cat) = 86 +/- 5 s(-1)). Progress of the reaction is monitored by spectrophotometric observation of decrease in NADPH concentration at 340 nm in a coupled enzyme assay with L-glutamate dehydrogenase (EC 1.4.1.4). Stereochemically pure la was synthesized as its trilithium salt by ene reaction of methyl glyoxylate with methyl N-succinyl-L-allylglycinate (4a) followed by hydrogenation of the double bond, Dess-Martin oxidation of the alcohol, and careful lithium hydroxide hydrolysis. Similar approaches allowed synthesis of a series of substrate analogues 1b-g having different N-acyl substituents, as well as derivatives missing the carboxyl group or the amide functionality (13 and 17, respectively). Compounds lacking the keto functionality (18a, 18c, and 19) were also prepared. Assay of DAP-AT shows that the enzyme has quite strict requirements for substrate recognition, but it will accept compounds with an aromatic ring in place of the terminal succinyl carboxyl group in la (e.g. N-Cbz-alpha-amino-epsilon-ketopimelic acid (1c)). Reaction of substrates 1a,c with hydrazine hydrate followed by NaCNBH3 reduction gives 2-(N-(succinylamino))-(20a) and 2-(N-Cbz-amino)-6-hydrazinoheptane-1,7-dioic acids (20c), respectively. These are the most potent slow-binding inhibitors of any DAP-metabolyzing enzyme reported so far (K-i* for DAP-AT : 20a is 22 +/- 4 nM; 20c is 54 +/- 9 nM).