Inhibition of alanine racemase by alanine phosphonate: detection of an imine linkage to pyridoxal 5'-phosphate in the enzyme-inhibitor complex by solid-state 15N nuclear magnetic resonance.
Inhibition of alanine racemase from the Gram-positive bacterium Bacillus stearothermophilus by (1-aminoethyl) phosphonic acid (Ala-P) proceeds via a two-step reaction pathway in which reactivation occurs very slowly. In order to determine the mechanism of inhibition, we have recorded low-temperature, solid-state 15N NMR spectra from microcrystals of the [15N]Ala-P-enzyme complex, together with spectra of a series of model compounds that provide the requisite database for the interpretation of the 15N chemical shifts. Proton-decoupled spectra of the microcrystals exhibit a line at approximately 150 ppm, which conclusively demonstrates the presence of a protonated Ala-P-PLP aldimine and thus clarifies the structure of the enzyme-inhibitor complex. We also report the pH dependence of Ala-P binding to alanine racemase.
Copié
V
V
Francis Bitter National Magnet Laboratory, Massachusetts Institute of Technology, Cambridge 02139.
Faraci
W S
WS
Walsh
C T
CT
Griffin
R G
RG
D000411:Alanine Racemase / Q000037:antagonists & inhibitors*; D000591:Amino Acid Isomerases / Q000037:antagonists & inhibitors*; D000615:Aminoethylphosphonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology*; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D063065:Organophosphonates; D009943:Organophosphorus Compounds / Q000494:pharmacology*; D011732:Pyridoxal Phosphate / Q000378:metabolism*
D063065:Organophosphonates; D009943:Organophosphorus Compounds; D011732:Pyridoxal Phosphate; C047639:1-(aminoethyl)phosphonic acid; D000615:Aminoethylphosphonic Acid; D000591:Amino Acid Isomerases; D000411:Alanine Racemase