Electron transfer in the substrate-dependent suicide inactivation of lysine 5,6-aminomutase.

The lysine 5,6-aminomutase (5,6-LAM) purified from Clostridium sticklandii was found to undergo rapid inactivation in the absence of the activating enzyme E(2) and ATP. In the presence of substrate, inactivation was also seen for the recombinant 5,6-LAM. This adenosylcobalamin-dependent enzyme is postulated to generate cob(II)alamin and the 5'-deoxyadenosyl radical through enzyme-induced homolytic scission of the Co-C bond. However, the products cob(III)alamin and 5'-deoxyadenosine were observed upon inactivation of 5,6-LAM. Cob(III)alamin production, as monitored by the increase in A(358), proceeds at the same rate as the loss of enzyme activity, suggesting that the activity loss is related to the adventitious generation of cob(III)alamin during enzymatic turnover. The cleavage of adenosylcobalamin to cob(III)alamin is accompanied by the formation of 5'-deoxyadenosine at the same rate, and the generation of cob(III)alamin proceeds at the same rate both aerobically and anaerobically. Suicide inactivation requires the presence of substrate, adenosylcobalamin, and PLP. We have ruled out the involvement of either the putative 5'-deoxyadenosyl radical or dioxygen in suicide inactivation. We have shown that one or more reaction intermediates derived from the substrate or/and the product, presumably a radical, participate in suicide inactivation of 5,6-LAM through electron transfer from cob(II)alamin. Moreover, L-lysine is found to be a slowly reacting substrate, and it induces inactivation at a rate similar to that of D-lysine. The alternative substrate beta-lysine induces inactivation at least 25 times faster than DL-lysine. The inactivation mechanism is compatible with the radical isomerization mechanism proposed to explain the action of 5,6-LAM.