SYNTHESIS AND EVALUATION OF NOVEL SUBSTRATES AND INHIBITORS OF N-SUCCINYL-LL-DIAMINOPIMELATE AMINOTRANSFERASE (DAP-AT) FROM ESCHERICHIA-COLI

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 ne ar homogeneity (1500-fold) in five steps from wild type Escherichia co il ATCC 9637. This pyridoxal phosphate (PLP) dependent enzyme has a mo lecular weight of 39.9 kDa, appears to form an active homodimer, and u ses 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 spect rophotometric 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 e ne reaction of methyl glyoxylate with methyl N-succinyl-L-allylglycina te (4a) followed by hydrogenation of the double bond, Dess-Martin oxid ation of the alcohol, and careful lithium hydroxide hydrolysis. Simila r 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 al so prepared. Assay of DAP-AT shows that the enzyme has quite strict re quirements for substrate recognition, but it will accept compounds wit h an aromatic ring in place of the terminal succinyl carboxyl group in la (e.g. N-Cbz-alpha-amino-epsilon-ketopimelic acid (1c)). Reaction o f substrates 1a,c with hydrazine hydrate followed by NaCNBH3 reduction gives 2-(N-(succinylamino))-(20a) and 2-(N-Cbz-amino)-6-hydrazinohept ane-1,7-dioic acids (20c), respectively. These are the most potent slo w-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).