Partial purification and specificity studies of the D-glutamate-adding and D-alanyl-D-alanine-adding enzymes from Escherichia coli K12.
The D-glutamate-adding and D-alanyl-D-alanine-adding enzymes from Escherichia coli were partially purified by fast protein liquid chromatography on an anion exchanger. Their relative molecular masses, determined by gel filtration on Superose 12, were 54,000 +/- 2000 and 51,000 +/- 2000, respectively. In order to investigate the specificity of these ligases, several compounds derived from their respective nucleotide substrates were prepared. In the case of the D-Glu-adding enzyme, DDP-MurNAc-L-Ala (DDP = dihydrouridine 5'-diphosphate) and P1-MurNAc-L-Ala were substrates of the reaction. In the case of the D-Ala-D-Ala-adding enzyme, only DDP-MurNAc-L-Ala-D-Glu(-meso-A2pm) was a substrate; P1-MurNAc-L-Ala-D-Glu(-meso-A2pm) was neither a substrate nor an inhibitor. Concerning the amino acid site of the D-Glu-adding enzyme, even closely related analogues of D-glutamate hardly inhibited the reaction.
Michaud
C
C
Blanot
D
D
Flouret
B
B
Van Heijenoort
J
J
D002845:Chromatography / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D010453:Peptide Synthases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D013379:Substrate Specificity
D010453:Peptide Synthases; C019638:D-alanylalanine synthetase; C053379:UDP-N-acetylmuramoylalanine-D-glutamate ligase