title: Biochemical characterization of a phosphinate inhibitor of Escherichia coli MurC.
abstract: The bacterial UDP-N-acetylmuramyl-L-alanine ligase (MurC) from Escherichia coli, an essential, cytoplasmic peptidoglycan biosynthetic enzyme, catalyzes the ATP-dependent ligation of L-alanine (Ala) and UDP-N-acetylmuramic acid (UNAM) to form UDP-N-acetylmuramyl-L-alanine (UNAM-Ala). The phosphinate inhibitor 1 was designed and prepared as a multisubstrate/transition state analogue. The compound exhibits mixed-type inhibition with respect to all three enzyme substrates (ATP, UNAM, Ala), suggesting that this compound forms dead-end complexes with multiple enzyme states. Results from isothermal titration calorimetry (ITC) studies supported these findings as exothermic binding was observed under conditions with free enzyme (K(d) = 1.80-2.79 microM, 95% CI), enzyme saturated with ATP (K(d) = 0.097-0.108 microM, 95% CI), and enzyme saturated with the reaction product ADP (K(d) = 0.371-0.751 microM, 95% CI). Titrations run under conditions of saturating UNAM or the product UNAM-Ala did not show heat effects consistent with competitive compound binding to the active site. The potent binding affinity observed in the presence of ATP is consistent with the inhibitor design and the proposed Ordered Ter-Ter mechanism for this enzyme; however, the additional binding pathways suggest that the inhibitor can also serve as a product analogue.
authors.affiliation: Department of Biochemistry, Cancer and Infection Research Area, AstraZeneca R&D Boston, Waltham, Massachusetts 02451, USA.
authors.forename: S
authors.initials: S
authors.lastname: Marmor
authors.forename: C P
authors.initials: CP
authors.lastname: Petersen
authors.forename: F
authors.initials: F
authors.lastname: Reck
authors.forename: W
authors.initials: W
authors.lastname: Yang
authors.forename: N
authors.initials: N
authors.lastname: Gao
authors.forename: S L
authors.initials: SL
authors.lastname: Fisher
mesh_terms: D001483:Base Sequence; D002151:Calorimetry; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D010453:Peptide Synthases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010721:Phosphinic Acids / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D017931:DNA Primers; D010721:Phosphinic Acids; D010453:Peptide Synthases; C023229:UDP-N-acetylmuramoyl-alanine synthetase