Radical triplets and suicide inhibition in reactions of 4-thia-D- and 4-thia-L-lysine with lysine 5,6-aminomutase.

Lysine 5,6-aminomutase (5,6-LAM1) participates in the fermentation of l- or d-lysine as carbon and nitrogen sources in anaerobic bacteria (1). Anaerobic fermentation of l-lysine proceeds efficiently as in Figure 1, starting with conversion to l-β-lysine by 2,3-LAM, a SAM and PLP-dependent enzyme. 5,6-LAM then converts l-β-lysine into l-3,5-DAH, a molecule poised for dehydrogenation and β-oxidation. Fermentation of d-lysine in Figure 1 begins with conversion to d-2,5-DAH by 5,6-LAM and proceeds to the formation of acetate and butyrate (1). Figure 1 Metabolism of lysine in anaerobic bacteria. 5,6-LAM is an adenosylcobalamin- and PLP-dependent enzyme that catalyzes the interconversion of d- or l-lysine with d- or l-2,5-DAH or of l-β-lysine with l-3,5-DAH (1-8). The mechanism of action of 2,3-LAM is well worked out, and the structure of the enzyme is fully compatible with the spectroscopic and chemical evidence supporting the mechanism (9,10). The 2,3-LAM mechanism inspires the hypothetical chemical mechanism for 5,6-LAM shown in Scheme 1 (2,4,9), wherein the 5′-deoxyadenosyl radical from adenosylcobalamin initiates the chemistry by abstracting a C5(H) from lysine to generate the substrate-related radical 2, which is bound as the Ne-aldimine to PLP. Radical isomerization analogous to that in 2,3-LAM leads through the aziridincarbinyl intermediate 3 to the product-related radical 4, which is quenched by hydrogen transfer from 5′-deoxyadenosine. In contrast to 2,3-LAM, little experimental evidence bearing on the mechanism of action of 5,6-LAM is available, apart from the mediation of hydrogen transfer by the 5′-deoxyadenosyl moiety of adenosylcobalamin (7). The X-ray crystal structure of 5,6-LAM raises questions regarding coordination in the actions of PLP and adenosylcobalamin (11). Scheme 1 5,6-LAM is a heterotetrameric protein composed of α- and β-subunits (αβ)2. In the available structure, illustrated in Figure 2 with cobalamin, 5′-deoxyadenosine and PLP as ligands, the α-subunit incorporates a TIM barrel and the β-subunit a Rossman domain. Adenosylobalamin binds in a base-off mode, with most interactions to the β-subunit, which projects the 5′-deoxyadenosyl moiety toward the β–barrel of the α–subunit. The major binding contacts of PLP are to the α–subunit, but the β-subunit binds the carboxaldehyde group of PLP as an internal aldimine with Lysβ144 (4,11). The 24 A separation between 5′-deoxyadenosine and PLP in the structure is too great to represent an active conformation that would allow a substrate to interact chemically with both adenosylcobalamin and PLP. Figure 2 Structure of 5,6-LAM and relative locations of adenosylcobalamin and PLP. Spectroscopic experiments show that other adenosylcobalamin-dependent enzymes facilitate the transient and reversible homolytic cleavage of the Co—C5′ bond in adenosylcobalamin to form cob(II)alamin. The resultant 5′-deoxyadenosyl radical initiates catalysis by abstracting a hydrogen atom from the cognate substrate (12-14). Limited evidence for homolytic scission of the Co—C5′ bond is available for 5,6-LAM. Cob(II)alamin is not observable as an intermediate in the steady state with any substrate. The only reported cleavages of the Co—C5′ bond by 5,6-LAM are the formation of cob(III)alamin during suicide inactivation of the enzyme by substrates (2), and the EPR spectroscopic observation of cob(II)alamin in a reaction with the substrate analog 4-thia-l-lysine (15). EPR spectroscopy is employed in research on the mechanisms of enzymes catalyzing radical reactions, allowing structural assignments to intermediates that are detectable by EPR (16-20). No radical can be detected in the reactions of 5,6-LAM with the natural substrates d-lysine, l-lysine, or l-β-lysine. In this report, we present the results of studies of the reaction of 5,6-LAM with 4-thia-d- and 4-thia-l-lysine. These molecules are structurally similar to d- and l-lysine but have special chemical properties that facilitate the spectroscopic observation of radicals related in structure to possible catalytic intermediates. We also present spectrophotometric evidence for the reaction of 4-thia-d- and 4-thia-l-lysine in cleaving the Co—C5′ bond of adenosylcobalamin to cob(II)alamin and 5′-deoxyadenosine. The results support catalysis of amino group migration by way of the radical mechanism in Scheme 1.