INHIBITION OF ALANINE RACEMASE BY ALANINE PHOSPHONATE - DETECTION OF AN IMINE LINKAGE TO PYRIDOXAL 5'-PHOSPHATE IN THE ENZYME-INHIBITOR COMPLEX BY SOLID-STATE N-15 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 specra of series of model compounds that provide the requisite database for the intepretation of the 15N chemical shifts. Proton-decoupled spectra of the microcrystals exhibit a line at .apprx.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.