Inhibition of alanine racemase by alanine phosphonate: detection of an imine linkage to pyridoxal 5'-phosphate in the enzyme-inhibitor complex by solid-state nitrogen-15 nuclear magnetic resonance

Abstract

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.

Carbon and Nitrogen Chemical Shifts of Solid State Enzymes

“…Solid state NMR data indicate that this compound forms a protonated imine with PLP at the active site, thus confirming that inhibition proceeds by the same mechanism as has been proposed for catalysis. 32 In bacteriorhodopsin the Schiff base linkage between the retinal and a lysine side chain has been demonstrated by 15 N tensor analysis, 33 and it is known now to be protonated in all states studied with the exception of the 'M' photointermediate (Scheme 9). The effects of the counterion on the chemical shift appear in the middle and deshielded only tensor elements (δ 22 and δ 11 ) in a fairly systematic fashion, indicating that tensorial information will be useful for assigning the causes of changes in shift for this case.…”

Biological Activity of Aminophosphonic Acids

“…Phosphonic analogue 3 of alanine is a potent inhibitor of a racemase from Gram-positive bacteria. [17][18][19] The aminophosphonate binds with pyridoxal5'-phosphate in a manner identical to alanine. The formation of inhibitor-coenzyme aldimine is followed by a change in the enzyme conformation resulting in tight binding of the inhibitor.…”

The Alanine Racemase of Mycobacterium smegmatis Is Essential for Growth in the Absence of d -Alanine

“…Because of its importance to global health, there is great interest in the identification and development of targets for antimycobacterial drug design. One such target of significant historical interest is alanine racemase (5,14,22). Alanine racemase is a pyridoxal phosphate-containing homodimeric enzyme that catalyzes the conversion of L-alanine to D-alanine, a key building block in the biosynthesis of the peptidoglycan layer in bacterial cell walls.…”