Assay and properties of diaminopimelate epimerase from Bacillus megaterium.
1. Diaminopimelate epimerase from a soluble extract of Bacillus megaterium N.C.I.B. 7581 was purified about 25-fold by fractionation with ammonium sulphate and chromatography on calcium phosphate gel-cellulose. The product was impure but was unstable on further purification. 2. Quantitative assay methods for the enzyme were devised in which meso- or ll-diaminopimelic acid may be the substrate. 3. Between 25 degrees and 45 degrees at pH7.0 enzyme action leads to an equilibrium mixture containing 65% meso-isomer and 35% ll-isomer. 4. The initial rate of epimerization was 2-3 times as fast with ll-diaminopimelic acid as substrate as with the meso-isomer; a number of other amino acids were not racemized by the enzyme. The Michaelis constants at 37 degrees were 6.7mm (ll-isomer) and 100mm (meso-isomer); with both substrates enzyme activity was maximal at pH7-8. The relative rates of epimerization of ll-diaminopimelic acid at 25 degrees , 37 degrees and 45 degrees were 0.77:1.00:1.15. 5. A thiol compound (of which 2,3-dimercaptopropan-1-ol was the most effective) was needed as an activator of the purified enzyme. 6. Carbonylbinding reagents and several other compounds did not inhibit diaminopimelate epimerase. 7. Pyridoxal phosphate did not stimulate enzymic activity even in preparations that had been almost completely freed of derivatives of vitamin B(6) (as shown by microbiological assay).
D000596:Amino Acids; D001410:Bacillus megaterium / Q000201:enzymology*; D001681:Biological Assay; D011232:Chemical Precipitation; D002845:Chromatography; D002850:Chromatography, Gel; D004112:Dimercaprol; D004355:Drug Stability; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007535:Isomerases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D007700:Kinetics; D008365:Manometry; D010867:Pimelic Acids; D011732:Pyridoxal Phosphate; D011736:Pyridoxine; D013237:Stereoisomerism; D013696:Temperature
D000596:Amino Acids; D010867:Pimelic Acids; D004112:Dimercaprol; D011732:Pyridoxal Phosphate; D007535:Isomerases; D011736:Pyridoxine