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

Abstract

Lysine 5,6-aminomutase (5,6-LAM) catalyzes the interconversions of D- or L-lysine and the corresponding enantiomers of 2,5-diaminohexanoate, as well as the interconversion of L-beta-lysine and l-3,5-diaminohexanoate. The reactions of 5,6-LAM are 5'-deoxyadenosylcobalamin- and pyridoxal-5'-phosphate (PLP)-dependent. Similar to other 5'-deoxyadenosylcobalamin-dependent enzymes, 5,6-LAM is thought to function by a radical mechanism. No free radicals can be detected by electron paramagnetic resonance (EPR) spectroscopy in reactions of 5,6-LAM with D- or L-lysine or with L-beta-lysine. However, the substrate analogues 4-thia-L-lysine and 4-thia-D-lysine undergo early steps in the mechanism to form two radical species that are readily detected by EPR spectroscopy. Cob(II)alamin and 5'-deoxyadenosine derived from 5'-deoxyadenosylcobalamin are also detected. The radicals are proximal to and spin-coupled with low-spin Co(2+) in cob(II)alamin and appear as radical triplets. The radicals are reversibly formed but do not proceed to stable products, so that 4-thia-D- and L-lysine are suicide inhibitors. Inhibition attains equilibrium between the active Michaelis complex and the inhibited radical triplets. The structure of the transient 4-thia-L-lysine radical is analogous to that of the first substrate-related radical in the putative isomerization mechanism. The second, persistent radical is more stable than the transient species and is assigned as a tautomer, in which a C6(H) of the transient radical is transferred to the carboxaldehyde carbon (C4') of PLP. The persistent radical blocks the active site and inhibits the enzyme, but it decomposes very slowly at </=1% of the rate of formation to regenerate the active enzyme. Fundamental differences between reversible suicide inactivation by 4-thia-D- or L-4-lysine and irreversible suicide inactivation by D- or L-lysine are discussed. The observation of the transient radical supports the hypothetical isomerization mechanism.

Scheme 1

Scheme 2

Scheme 3

Figure 1

Metabolism of lysine in anaerobic bacteria.

Figure 2

Structure of 5,6-LAM and relative locations of adenosylcobalamin and PLP. The structure is of 5,6-LAM with PLP, 5′-deoxyadenosine and cobalamin as ligands (11). This image was created by H. Adam Steinberg from PDB ID 1XRS.

Figure 3

Isotope edited EPR spectra of the transient radical with 4-thia-l-lysine. EPR spectra at 9.1 GHz of the transient radical with 100 μM activated 5,6-LAM, 100 μM adenosylcobalamin, and 100 μM PLP reacting with 20 mM 4-thia-l-lysine without and with ²H, ¹³C, or ¹⁵N-labeling and freeze quenched at 10 s. In this spectrum, g =2.0 corresponds to 3261 G.

Figure 4

Isotope edited EPR spectra of the persistent radical with 4-thia-l-lysine. Spectra at 9.1 Ghz were obtained as in Figure 1 but after freeze quenching at 10 min. In this spectrum, g =2.0 corresponds to 3261 G.

Figure 5

EPR spectral simulations of transient and persistent radicals in the reaction of l-4-thialysine. The upper spectrum is of the transient radical (—) and the simulation (- - -). Parameters used in the calculated spectrum: Co²⁺ g-tensor, 2.32, 2.22, 2.00; radical g-tensor, 2.009, 2.003, 2.002; ZFS parameters, D = −75 G, E = 0 G; exchange coupling constant, J = 4 × 10³ G; ⁵⁹Co-hyperfine tensor (G), 10, 7, 112; isotropic ¹⁴N-hyperfine splitting, 19 G. Euler angles relating the interspin vector to the Co²⁺ g-axis: ζ = 43°, η = 0°, and ξ = 78°. A uniform line width of 6 G was used in the calculation. The lower spectrum is of the persistent radical (—) and the simulation (- - -). Parameters used in the calculated spectrum: Co²⁺ g-tensor, 2.32, 2.20, 2.00; radical g-tensor, 2.009, 2.004, 2.002; ZFS parameters, D = −20 G, E = 0 G; exchange coupling constant, J = 525 G; ⁵⁹Co-hyperfine tensor (G), 10, 7, 112; isotropic ¹⁴N-hyperfine splitting, 19 G. Euler angles relating the interspin vector to the Co²⁺ g-axis: ζ = 60°, η = 0°, and ξ = 35°. A uniform line width of 8 G was used in the calculation. In these spectra, g =2.0 corresponds to 3261 G.

Figure 6

Time course of inhibition and radical- and 5′-deoxyadenosine-formation. Reaction of 5,6-LAM (50 μM), 120 μM PLP, 120 μM [adenine-¹⁴C]adenosylcobalamin at pH 8.5 (EPPS buffer) was initiated with 20 mM 4-thia-d-lysine. □, Time course for increasing radical spin (μM); ●, time course for the formation of 5′-deoxy[8-¹⁴C]adenosine (μM); inset, enzyme activity.

Figure 7

Spectrophotometry in the reaction of 5,6-LAM with 4-thia-l-lysine. The anaerobic solution contained 23 μM 5,6-LAM, 25 μM PLP, and 23 μM adenosylcobalamin in 100 mM K⁺EPPS buffer at pH 8.5, with 4-thia-l-lysine in a side arm. The solid-line spectra are: (—), 5,6-LAM-adenosylcobalamin-PLP before addition of 4-thia-l-lysine; (—), within 10 s of addition of 4-thia-l-lysine to 20 mM; (—), 30 min after addition of 4-thia-l-lysine. The dashed spectra are: (- - -), 5,6-LAM-adenosylcobalamin and (- - -), 5,6-LAM-cob(II)alamin after anaerobic photolysis of 5,6-LAM adenosylcobalamin. In the cleavage of adenosylcobalamin the 420 nm band is retained as that of the external PLP-4-thia-l-lysine aldimine.

Figure 8

Structures under consideration for the transient and persistent radicals. At the top is the structure of the 4-thia-analog of radical 2 in Scheme 1, the transient radical 6. The lower four are candidate structures for the persistent radical. The quinonoid radical 7 is the product of proton transfer from C6 of the 4-thialysyl side chain to N1 of PLP. The tautomerization radical 8 is the product of proton transfer from C6 of the 4-thialysyl side chain to C4′ of PLP. The transaldimination radical 9 is the product of aldimine transfer from N6 to N2 of the 4-thialysyl side chain. The aziridincarbinyl radical 10 is the 4-thia-analogue of the intermediate radical 3 in Scheme 1.