Metabolic pathway assay

Details Metabolic pathway assay Metabolic pathway assay

1997-09-24

1999-04-06

Leary, Louise N.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

435 23, 435 24, 435 32, 435 15, 435 21, 435 791, 435 18, 435 16, C12Q 100, C12Q 137, C12Q 118, C12Q 148

Patent

active

058916210

ABSTRACT:
An in vitro screening assay which identifies enzyme inhibitors and allows for the simultaneous assay of many enzymes. Enzyme, substrate, co-factor, etc. concentrations are optimized so that inhibitors of any one of the enzymes in the pathway are equally likely to be detected. Necessarily, the flux of substrate through each enzyme should be nearly the same during the assay, i.e., each of the enzyme catalyzed steps must be equally rate-limiting. Preferably, optimal assay conditions are predicted by computer modeling. Further, the pathway conditions are optimized through variation of enzyme, starting substrate, co-substrate and co-factor concentrations. A positive response is initially detected as a change in the amount of the product generated at the end of the enzyme cascade as compared to a standard. A sample producing a positive result can be deconvoluted.

REFERENCES:
Athel Cornish-Bowden "Metabolic Control Analsys in Theory and Practice," Adv. Mol. Cell. Biol., vol. 11, pp. 21-64 (1995).
Tanner, et al., "Phosphinate Inhibitors of the d-Glutamic Acid-Adding Enzyme of Peptidoglycan Biosynthesis," J. Org. Chem., vol. 61, pp. 1756-1760 (1996).
Falk, et al., "Biochemical Evidence for the Formation of a Covalent Linkage between UDP-Acetylmuramate and ATP in the Escherichia coli UDP-N-Acetylmuramate:L-Alanine Ligase-Catalyzed Reaction," Biochemistry, vol. 35, pp. 1417-1422 (1996).
Jin, et al., "Structural Studies of Escherichia coli UDP-N-Acetylmuramate:L-Alanine Ligase," Biochemistry, vol. 35, pp. 1423-1431 (1996).
Auger, et al., "Synthesis of N-Acetylmuranic Acid Derivatives as Potential Inhibitors of the D-Glutamic Acid-Adding Enzyme," J. prakt. Chem., vol. 337, pp. 351-357 (1995).
Pratviel-Sosa, et al., "Over-production, purification and properties of the uridine diphosphate N-acetylmuramoyl-L-Alanine: D-glutamate ligase from Escherichia coli," Eur. J. Bio. Chem., vol. 202, pp. 1169-1176 (1991).
Michaud, et al., "Partial purification and specificity studies of the D-glutamate-adding and d-alanyl-D-alanine-adding enzymes from Escherichia coli K12," Eur. J. Biochem., vol. 166, pp. 631-637 (1987).
LeRoux et al., "Synthesis of new peptide inhibitors of the mesol-diaminopimelate-adding enzyme," Eur. J. Med. Chem., vol. 27, pp. 899-907 (1992).
Pratviel-Sosa, et al., "Effect of various analogues of D-glutamic acid on the D-glutamate-adding enzyme from Escherichia coli," FEMS, Microbiol. Letters, vol. 115, pp. 223-228 (1994).
Michaud et al., "Over-production, purification and properties of the uridine-diphosphate-N-acetylmuramoyl-L-alanyl-D-glutamate: meso-2,6-diaminopimelate ligase from Escherichia coli, " Eur. J. Biochem. vol. 194, pp. 853-861 (1990).
Liger et al., "Over-production purification and properties of the uridine-diphosphate-N-acetylmuramate:L-alanine ligase from Escherichia coli," Eur. J. Biochem., vol. 230, pp. 80-87 (1995).
Gubler et al., "Overexpression, Purification, and Characterization of UDP-N-Acetylmuramyl:L-Alanine Ligase from Escherichia coli," Journal of Bacteriology, Feb. 1996, pp. 906-910 (1996).
Abo-Ghalia, et al., "Specificity of the uridine-diphosphate-N-acetylmuramyl-L-alanyl-D-glutamate:meso-2, 6-diaminopimelate synthetase from Escherichia coli," Eur. J. Biochem., vol. 53, pp. 81-87 (1985).
Bugg and Walsh, "Intracellular Steps of Bacterial Cell Wall Peptidoglycan Biosynthesis: Enzymology, Antibiotics, and Antibiotic Resistance," Natural Product Reports (1992).
Presentation by Dr. D. Pompliano, Apr. 23, 1996, to University of Minnesota Departmental Staff Meeting.
Hakes & Dixon "New Vectors for High Level Expression of Recombinant Proteins in Bacteria," Analy. Biochem., vol. 202, pp. 293-298 (1992).
Reddy, et al., Mechanistic Analysis of UDP-N-acetylmuramyl:L-alamine Ligase from Escherichia coli presented at Enzyme Mechamism 15th Conference, Naple, Florida, Jan. 4, 1997-Jan. 8, 1997.

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