Evidence for Conformational Movement and Radical Mechanism in the Reaction of 4-Thia-l-lysine with Lysine 5,6-Aminomutase

Abstract

We demonstrate that the steady state reaction of lysine 5,6-aminomutase with substrate analogue 4-thia-l-lysine generates a radical intermediate, which accumulates in the enzyme to an electron paramagnetic resonance (EPR) detectable level. EPR line width narrowing of approximately 1 mT due to [4'-(2)H] labeling of the pyridoxal-5'-phosphate (PLP), an isotropic hyperfine coupling of 40 MHz for the proton at C4' of PLP derived from (2)H electron nuclear double resonance (ENDOR) measurement, and spin density delocalization onto the (31)P of PLP realized from observations of the (31)P ENDOR signal provide unequivocal identification of the radical as a substrate-PLP-based species. X- and Q-band EPR spectra fittings demonstrate that this radical is spin coupled with the low spin Co(2+) in cob (II) alamin and the distance between the two species is about 10 A. These results provide direct evidence for the active site motion upon substrate binding, bringing the adenosylcobalamin to the proximity of substrate-PLP for subsequent H-atom abstraction and for the notion that lysine 5,6-aminomutase functions by a radical mechanism. Observation of (2)H-ENDOR signal also provides a reliable hyperfine coupling constant for future comparison with quantum-mechanical-based calculations to gain further insight into the molecular structure of this steady state radical intermediate.

Radical Triplets and Suicide Inhibition in Reactions of 4-Thia-d- and 4-Thia-l-lysine with Lysine 5,6-Aminomutase

“…The lack of complete conversion of the enzyme to this radical (see Figure 6) and the absence of a long wavelength absorbance (see Figure 7) are inconsistent with the quinonoid form. In addition, the magnitude of the PLP-C4′(H) hyperfine coupling calculated for the quinonoid radical 7 is smaller than the observed value (Table 2) (15). …”

“…Reaction of 4-thia- d -lysine leads to analogous radicals on a faster time scale (Supplemental Information). Deuterium labeling in the carboxaldehyde group of PLP (C4′) narrows the signal (15), clearly showing hyperfine coupling of PLP-C4′–H with the unpaired electron in the persistent species.…”

“…The electronic structure of the PLP-portion of this radical is consistent with the increased absorbance at 340 nm, the decreased intensity at 420 nm, and the isosbestic point in Figure 7. The electronic structure also accounts for the magnitude of the hyperfine coupling of PLP-C4′(H) (Table 2) (15). The calculated isotropic coupling constant of 44.7 MHz is similar to the experimental value of 40 MHz (15).…”

4′-CyanoPLP presents better prospect for the experimental detection of elusive cyclic intermediate radical in the reaction of lysine 5,6-aminomutase

“…In the case of 4,5-OAM, the evidence for radical mechanism was obtained using a substrate homologue [9]. Identification of corresponding S Å in the reaction of 5,6-LAM has been reported recently [10,11] using substrate analogue 4-thialysine. The characterization of that was confirmed by electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopic studies [12] on isotopeedited substrate analogues, 4-thia-[5-13 C]lysine [13] and 4-thia-[6-13 C]lysine [14].…”

5-Fluorolysine as alternative substrate of lysine 5,6-aminomutase: A computational study

“…Additionally, they proposed that the reaction of 4-thialysine with 5,6-LAM would accumulate the corresponding substrate related radical to required concentration for EPR detection. In fact, evidence for the radical mechanism was recently reported by using substrate analogue 4-thialysine [27,28]. Tang et al reported the observation of two different radicals and proposed the presence of transient radical, which is the corresponding substrate related radical, and persistent radical [28].…”