Details
| Original language | English |
|---|---|
| Pages (from-to) | 7449-7460 |
| Number of pages | 12 |
| Journal | Journal of the American Chemical Society |
| Volume | 118 |
| Issue number | 32 |
| Publication status | Published - 14 Aug 1996 |
Abstract
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 coli 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-α-amino-ε-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 1a 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 gloup in 1a (e.g. N-Cbz-α-amino-ε-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-metabolizing enzyme reported so far (K(i)* for DAP-AT: 20a is 22 ± 4 nM, 20c is 54 ± 9 nM).
ASJC Scopus subject areas
- Chemical Engineering(all)
- Catalysis
- Chemistry(all)
- Biochemistry, Genetics and Molecular Biology(all)
- Biochemistry
- Chemical Engineering(all)
- Colloid and Surface Chemistry
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In: Journal of the American Chemical Society, Vol. 118, No. 32, 14.08.1996, p. 7449-7460.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Synthesis and evaluation of novel substrates and inhibitors of N-succinyl-LL-diaminopimelate aminotransferase (DAP-AT) from Escherichia coli
AU - Cox, Russell J.
AU - Sherwin, William A.
AU - Lam, Lister K.P.
AU - Vederas, John C.
PY - 1996/8/14
Y1 - 1996/8/14
N2 - 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 coli 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-α-amino-ε-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 1a 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 gloup in 1a (e.g. N-Cbz-α-amino-ε-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-metabolizing enzyme reported so far (K(i)* for DAP-AT: 20a is 22 ± 4 nM, 20c is 54 ± 9 nM).
AB - 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 coli 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-α-amino-ε-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 1a 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 gloup in 1a (e.g. N-Cbz-α-amino-ε-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-metabolizing enzyme reported so far (K(i)* for DAP-AT: 20a is 22 ± 4 nM, 20c is 54 ± 9 nM).
UR - http://www.scopus.com/inward/record.url?scp=0029782572&partnerID=8YFLogxK
U2 - 10.1021/ja960640v
DO - 10.1021/ja960640v
M3 - Article
AN - SCOPUS:0029782572
VL - 118
SP - 7449
EP - 7460
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
SN - 0002-7863
IS - 32
ER -