Purification and properties of diaminopimelic acid epimerase from Escherichia coli.
Diaminopimelic acid epimerase was purified from Escherichia coli. The enzyme is a monomer of Mr = 34,000. Diaminopimelic acid epimerase is not a pyridoxal phosphate-dependent enzyme: there is no evidence for pyridoxal phosphate in the ultraviolet spectrum of the purified enzyme, and the epimerase is not inactivated by carbonyl reagents such as hydroxylamine and sodium borohydride. Exchange of the alpha-protons of the substrates, DL- and LL-diaminopimelic acid, with solvent accompanies epimerization; and exchange of 3H from solvent into diaminopimelic acid gives 3H primarily (80-90%) in the product isomer, regardless of whether the DL- or LL-isomer is substrate. From these results it is concluded that the epimerase utilizes a two-base mechanism for proton translocation. In these major aspects of its mechanism, diaminopimelic acid epimerase resembles proline racemase. It is argued that the relative values of the isotope fractionation factors for the two proton acceptor sites on the enzyme can be estimated from the isotope effects for the DL- and LL-isomers of diaminopimelic acid. The observed difference in the isotope effects predicts that one, but not both, of the proton acceptor sites is a thiol, and it is demonstrated that diaminopimelic acid epimerase has a single thiol which is necessary for activity and which reacts with iodoacetamide.
D000591:Amino Acid Isomerases; D002851:Chromatography, High Pressure Liquid; D003960:Diaminopimelic Acid / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology*; D007535:Isomerases / Q000302:isolation & purification*; D007536:Isomerism; D007700:Kinetics; D008433:Mathematics; D008954:Models, Biological; D008970:Molecular Weight; D019998:Racemases and Epimerases / Q000302:isolation & purification* / Q000378:metabolism; D014316:Tritium
D014316:Tritium; D003960:Diaminopimelic Acid; D007535:Isomerases; D019998:Racemases and Epimerases; D000591:Amino Acid Isomerases; C042032:diaminopimelate epimerase