Kinetic and biochemical analysis of the mechanism of action of lysine 5,6-aminomutase

Lysine 5,6-aminomutase (5,6-LAM) catalyzes the reversible and nearly isoenergetic transformations of d-lysine into 2,5-diaminohexanoate (2,5-DAH) and of l-β-lysine into 3,5-diaminohexanoate (3,5-DAH). The activity of 5,6-LAM depends on pyridoxal-5′-phosphate (PLP) and adenosylcobalamin. The currently postulated multistep mechanism involves at least 12 steps, two of which involve hydrogen transfer. The deuterium kinetic isotope effects on kcat and kcat/Km have been found to be 10.4 ± 0.3 and 8.3 ± 1.9, respectively, in the reaction of dl-lysine-3,3,4,4,5,5,6,6-d8. The corresponding isotope effects for reaction of dl-lysine-4,4,5,5-d4 are 8.5 ± 0.7 and 7.1 ± 1.2, respectively. Neither cob(II)alamin nor a free radical can be detected in the steady state by UV–Vis spectrophotometry or electron paramagnetic resonance (EPR) spectroscopy. Therefore, hydrogen abstraction from carbon-5 of the substrate side chain is rate limiting in the mechanism. dl-4-Oxalysine is an alternative substrate for 5,6-LAM. dl-4-Oxalysine reacts irreversibly because the product breaks down into ammonia, acetaldehyde, and dl-serine. The value of Km for the reaction of dl-4-oxalysine is lower than that for dl-lysine and that of kcat for dl-4-oxalysine is slightly lower than that for dl-lysine. As measured by values of kcat/Km, 5,6-LAM uses dl-4-oxalysine essentially as efficiently as the best substrates, d-lysine and l-β-lysine, and more efficiently than dl-lysine. dl-4-Oxalysine induces the same suicide inactivation by electron transfer as do the biological substrates. The putative substrate-related radical intermediate is not sufficiently stabilized by the nonbonding 4-oxa electrons to be detectable by EPR spectroscopy.