PMID:7873525
| Citation |
Ho, HT, Falk, PJ, Ervin, KM, Krishnan, BS, Discotto, LF, Dougherty, TJ and Pucci, MJ (1995) UDP-N-acetylmuramyl-L-alanine functions as an activator in the regulation of the Escherichia coli glutamate racemase activity. Biochemistry 34:2464-70 |
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| Abstract |
D-Glutamate is an essential component of the bacterial peptidoglycan. In Escherichia coli, the biosynthesis of D-glutamate is catalyzed by a glutamate racemase (encoded by the dga gene) and is regulated by UDP-N-acetylmuramyl-L-alanine [Doublet et al. (1994) Biochemistry 33, 5285], a bacterial peptidoglycan subunit precursor. Investigation was conducted to elucidate the interaction between the enzyme and its regulator. Whole and N-terminal truncated enzymes, encoded by individual constructs containing either a full-length or an N-terminal truncated dga gene, were evaluated. In the absence of the regulator, the purified whole enzyme showed a low-level basal racemase activity for which a Km value of 18.9 mM and a Vmax of 0.4 mumol/(min.mg) were determined, using D-glutamate as the substrate. Using the same substrate, in the presence of 6.5 microM UDP-N-acetylmuramyl-L-alanine, a Km value of 4.2 mM and a Vmax of 34 mumol/(min.mg) were measured. Similar kinetic parameters for the activated enzyme were obtained using L-glutamate as the substrate. The N-terminal truncated E. coli enzyme, with a 21 amino acid region removed, is similar in size to the Pediococcus pentosaceus glutamate racemase. Effects of the regulator on the full-length and the N-terminal truncated enzyme in the dialyzed cell lysate were compared. A host cell line, E. coli WM335 delta recA, containing a nonfunctional chromosomal dga gene was used to minimize the background interference. With 6.5 microM regulator added, the N-terminal truncated enzyme displayed a loss of more than 80% of the activity compared to the full-length enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) |
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| Keywords |
Amino Acid Isomerases/genetics; Amino Acid Isomerases/isolation & purification; Amino Acid Isomerases/metabolism; Amino Acid Sequence; Base Sequence; Cloning, Molecular; DNA Primers/genetics; DNA, Bacterial/genetics; Enzyme Activation/drug effects; Escherichia coli/enzymology; Escherichia coli/genetics; Genes, Bacterial; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Pediococcus/enzymology; Pediococcus/genetics; Peptidoglycan/biosynthesis; Peptidoglycan/chemistry; Sequence Homology, Amino Acid; Substrate Specificity; Uridine Diphosphate N-Acetylmuramic Acid/analogs & derivatives; Uridine Diphosphate N-Acetylmuramic Acid/biosynthesis; Uridine Diphosphate N-Acetylmuramic Acid/chemistry; Uridine Diphosphate N-Acetylmuramic Acid/metabolism; Uridine Diphosphate N-Acetylmuramic Acid/pharmacology |
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