title: A new robust kinetic assay for DAP epimerase activity.
abstract: DAP epimerase is the penultimate enzyme in the lysine biosynthesis pathway. The most versatile assay for DAP epimerase catalytic activity employs a coupled DAP epimerase-DAP dehydrogenase enzyme system with a commercial mixture of DAP isomers as substrate. DAP dehydrogenase converts meso-DAP to THDP with concomitant reduction of NADP(+) to NADPH. We show that at high concentrations, accumulation of NADPH results in inhibition of DAPDH, resulting in spurious kinetic data. A new assay has been developed employing DAP decarboxylase that allows the reliable characterisation of DAP epimerase enzyme kinetics.
authors.affiliation: School of Chemistry, The University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Lilian
authors.initials: L
authors.lastname: Hor
authors.forename: Martin G
authors.initials: MG
authors.lastname: Peverelli
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Perugini
authors.forename: Craig A
authors.initials: CA
authors.lastname: Hutton
mesh_terms: D000591:Amino Acid Isomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048230:Corynebacterium glutamicum / Q000201:enzymology* / Q000235:genetics; D003960:Diaminopimelic Acid / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods / Q000592:standards*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000096:biosynthesis; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D009249:NADP; D003960:Diaminopimelic Acid; D000594:Amino Acid Oxidoreductases; C020032:diaminopimelate dehydrogenase; D002262:Carboxy-Lyases; C489812:LysA protein, E coli; D000591:Amino Acid Isomerases; C042032:diaminopimelate epimerase; D008239:Lysine
keywords: DAP; DAP decarboxylase; DAP dehydrogenase; DAP epimerase; DAPDC; DAPDH; Diaminopimelate biosynthesis; Kinetic assay; PLP; THDP; diaminopimelate; diaminopimelate decarboxylase; diaminopimelate dehydrogenase; ll-DAP; pyridoxal-5′-phosphate; tetrahydrodipicolinate