Radical Triplets and Suicide Inhibition in Reactions of 4-Thia-d - and 4-Thia-l -lysine with Lysine 5,6-Aminomutase
| Autor: | Perry A. Frey, Steven O. Mansoorabadi, George H. Reed, Kuo-Hsiang Tang |
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| Rok vydání: | 2009 |
| Předmět: |
Models
Molecular Time Factors Free Radicals Protein Conformation Stereochemistry Radical Substrate analog complex mixtures Biochemistry Article chemistry.chemical_compound medicine Cysteine Enzyme Inhibitors Intramolecular Transferases Bond cleavage Transcobalamins Deoxyadenosines Chemistry Electron Spin Resonance Spectroscopy Deuterium Exchange Measurement Substrate (chemistry) Stereoisomerism Adenosylcobalamin Homolysis Spectrophotometry Suicide inhibition Biocatalysis bacteria Quantum Theory Cobamides Anaerobic bacteria Porphyromonas gingivalis medicine.drug |
| Zdroj: | Biochemistry. 48:8151-8160 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi900828f |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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