Isotope effects for deuterium transfer and mutagenesis of Tyr187 provide insight into controlled radical chemistry in adenosylcobalamin-dependent ornithine 4,5-aminomutase.

Adenosylcobalamin-dependent ornithine 4,5-aminomutase (OAM) from Clostridium sticklandii utilizes pyridoxal 5′-phosphate (PLP) to interconvert d-ornithine to 2,4-diaminopentanoate via a multistep mechanism that involves two hydrogen transfer steps. Herein, we uncover features of the OAM catalytic mechanism that differentiate it from its homologue, the more catalytically promiscuous lysine 5,6-aminomutase. Kinetic isotope effects (KIEs) with dl-ornithine-3,3,4,4,5,5-d6 revealed a diminished Dkcat/Km of 2.5 ± 0.4 relative to a Dkcat of 7.6 ± 0.5, suggesting slow release of the substrate from the active site. In contrast, a KIE was not observed on the rate constant associated with Co–C bond homolysis as this step is likely “gated” by the formation of the external aldimine. The role of tyrosine 187, which lies planar to the PLP pyridine ring, was also investigated via site-directed mutagenesis. The 25- and 1260-fold reduced kcat values for Y187F and Y187A, respectively, are attributed to a slower rate of exte...