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L-a-lysyl-PLP radical (27) 28 L-yS-lysyl-PLP radical (29)

Scheme 1.6.7. Reaction mechanism for generation of (S)-b-lysine catalyzed by lysine 2,3-aminomutase.

L-a-lysyl-PLP radical (27) 28 L-yS-lysyl-PLP radical (29)

Scheme 1.6.7. Reaction mechanism for generation of (S)-b-lysine catalyzed by lysine 2,3-aminomutase.

The reaction mechanism outlined was confirmed by direct observation of the ¡-lysyl-PLP radical 29 by EPR methods [37]. A strong signal was detected in the EPR spectrum by incubation of lysine 2,3-aminomutase with a-lysine and SAM and subsequent freezing in the steady state with liquid N2. The presence of a ¡-lysine-

PLP radical was proved by application of [2-2H]lysine and [2-13C]lysine instead of lysine 24 to the reaction mixture [38]. In the first experiment the EPR signal was narrowed; in the second it was broadened. The involvement of PLP in the reaction was proven by ESEEM spectroscopy. By incubation of the aminomutase with lysine, SAM, and [4'-2H]PLP a prominent doublet centered at the Lamour frequency for 2H was recognized [39], in accordance with the structure of an external aldimine. These findings establish a new role for PLP in enzyme reactions - PLP facilitates the radical isomerization.

Other radicals could not be detected directly by EPR, because of their instability. By use of analogous molecules which stabilize these radicals, however, their existence was rationalized. Stabilization of an a-lysyl radical analog was achieved by use of 4-thialysine 32 [40] (Scheme 1.6.8), with trans-4,5-dehydrolysine [41] as substrate.

Scheme 1.6.8. Incubation of 4-thialysine with lysine 2,3-aminomutase generates 4-thialysyl-PLP radicals.

NHZ pLp N-PLP

4-thialysine (32) 4-thialysyl-PLP radical (33)

Scheme 1.6.8. Incubation of 4-thialysine with lysine 2,3-aminomutase generates 4-thialysyl-PLP radicals.

It was also possible to confirm by EPR the existence of the 5 '-deoxyadenosyl radical species by use of 3 ',4'-anhydroadenosylmethionine 34 as allyl analog of SAM [42] (Scheme 1.6.9).

3',4'-anhydroadenosylmethionine (34) methionine (15) 3',4'-anhydro-5'-deoxyadenosyl radical (35)

Scheme 1.6.9. Generation of the allyl analog 35 of the 5 '-deoxyadenosyl radical 31.

3',4'-anhydroadenosylmethionine (34) methionine (15) 3',4'-anhydro-5'-deoxyadenosyl radical (35)

Scheme 1.6.9. Generation of the allyl analog 35 of the 5 '-deoxyadenosyl radical 31.

This reaction mechanism seems not to be restricted to the lysine 2,3-aminomutase itself. The cobalamin-dependent lysine 5,6-aminomutase and the ornithine 4,5-aminomutase from C. sticklandii follow apparently the same reaction mechanism except that they need B12 instead of SAM as cofactor.

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