Irreversible inactivation of lactate racemase by sodium borohydride reveals reactivity of the nickel-pincer nucleotide cofactor.

The nickel-pincer nucleotide (NPN) cofactor discovered in lactate racemase from (LarA) is essential for the activities of racemases/epimerases in the highly diverse LarA superfamily. Prior mechanistic studies have established a proton-coupled hydride-transfer mechanism for LarA, but direct evidence showing that hydride attacks the C4 atom in the pyridinium ring of NPN has been lacking. Here, we show that sodium borohydride (NaBH) irreversibly inactivates LarA accompanied by a rapid color change of the enzyme. The altered ultraviolet-visible spectra during NaBH titration supported hydride transfer to C4 of NPN, and the concomitant Ni loss unraveled by mass spectrometry experiments accounted for the irreversible inactivation. High resolution structures of LarA revealed a substantially weakened C-Ni bond in the metastable sulfite-NPN adduct where the NPN cofactor is in the reduced state. These findings allowed us to propose a mechanism of LarA inactivation by NaBH that provides key insights into the enzyme-catalyzed reaction and sheds light on the reactivity of small molecule NPN mimetics.