title: 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.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, 1710 University Avenue, Madison, Wisconsin 53726, USA.
authors.forename: Kuo-Hsiang
authors.initials: KH
authors.lastname: Tang
authors.forename: Steven O
authors.initials: SO
authors.lastname: Mansoorabadi
authors.forename: George H
authors.initials: GH
authors.lastname: Reed
authors.forename: Perry A
authors.initials: PA
authors.lastname: Frey
mesh_terms: D055162:Biocatalysis; D003038:Cobamides / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D003839:Deoxyadenosines / Q000737:chemistry / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D005609:Free Radicals / Q000737:chemistry* / Q000378:metabolism; D019751:Intramolecular Transferases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D016966:Porphyromonas gingivalis / Q000201:enzymology; D011487:Protein Conformation; D011789:Quantum Theory; D013053:Spectrophotometry; D013237:Stereoisomerism; D013997:Time Factors; D014155:Transcobalamins / Q000737:chemistry / Q000378:metabolism
chemical_list: D003038:Cobamides; D003839:Deoxyadenosines; D004791:Enzyme Inhibitors; D005609:Free Radicals; D014155:Transcobalamins; C000204:S-2-aminoethyl cysteine; C033714:5'-deoxyadenosine; D019751:Intramolecular Transferases; C403167:D-lysine 5,6-aminomutase; C000913:cobamamide; D003545:Cysteine