Study of the reaction mechanism of the D-glutamic acid-adding enzyme from Escherichia coli.
The D-glutamic acid-adding enzyme of Escherichia coli, or MurD, was purified from an overproducing strain and a few aspects of its reaction mechanism were studied. The existence of a reactive cysteinyl residue was shown by the following experiments: (1) two thiol-modifying reagents, (5,5'-dithiobis)2-nitrobenzoic acid and 2-nitro-5-thiocyanobenzoic acid, inactivated the enzyme; (2) incubation with tetranitromethane led to inactivation and to the appearance of cysteic acid (not to 3-nitrotyrosine); (3) in each case, ATP or UDP-MurNAc-L-Ala (but not D-glutamic acid) protected the enzyme from inactivation. The existence of a reactive lysyl residue was shown by the action of 2,4,6-trinitrobenzenesulfonic acid, a reagent specific for lysyl residues present in phosphate-binding sites. The formation of an acyl phosphate intermediate was consistent with three types of results: (1) the molecular isotope exchange reaction, which took place only in the presence of phosphate, but which was not strictly dependent on the presence of ADP; (2) a release of phosphate, measured by the molybdate assay, observed when the enzyme was incubated with ATP and UDP-MurNAc-L-Ala (without D-glutamic acid); (3) the appearance of a new radioactive compound (besides ATP and Pi) after incubation for a few minutes with UDP-MurNAc-L-Ala and [gamma-32P]ATP. Finally, the fact that phosphinate 1 was a good inhibitor of the enzyme (IC50 = 0.7 microM) strongly suggested that a tetrahedral transition state follows the acyl phosphate in the reaction pathway.
Vaganay
S
S
URA 1131 du CNRS, Båtiment 432, Université de Paris-Sud, Orsay, France.
Tanner
M E
ME
van Heijenoort
J
J
Blanot
D
D
D002855:Chromatography, Thin Layer; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D018698:Glutamic Acid / Q000378:metabolism*; D007700:Kinetics; D010453:Peptide Synthases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010710:Phosphates / Q000737:chemistry; D013439:Sulfhydryl Reagents
D004791:Enzyme Inhibitors; D010710:Phosphates; D013439:Sulfhydryl Reagents; D018698:Glutamic Acid; D010453:Peptide Synthases; C053379:UDP-N-acetylmuramoylalanine-D-glutamate ligase