RESUMO
Methionine-γ-lyase (MGL) is a pyridoxal-5'-phosphate dependent enzyme found in bacteria and protozoa that catalyzes a variety of reactions, including the γ-elimination of L-methionine (L-Met). Here we report experimental kinetic data and density functional theory (DFT) computational data for the γ-elimination reaction of L-Met and several other substrate analogues by a recombinant MGL from P. gingivalis (MGL_Pg). UV-Visible spectrophotometry experiments revealed a heavily populated species with maximum absorbance at 478 nm during steady-state catalysis of L-Met, L-ethionine, L-methionine sulfone and L-homoserine, which we assign to a late crotonate intermediate formed after the γ-cleavage step in the reaction and thus common to all substrates. A more red-shifted (498 nm) species was observed during the reaction of L-homoserine lactone, which we assign to an early quinonoid intermediate with the aid of time-dependent self-consistent field calculations. Significant differences in both binding and the rate of turnover were observed for the substrates. MGL_Pg's highest catalytic efficiency was recorded for L-vinylglycine (kcat/Km = 6455 s-1 M-1), exceeding that of L-Met (kcat/Km = 4211 s-1 M-1), while L-Met sulfone displayed the largest turnover number (kcat = 1638 min-1). A direct correlation between experimental kcat values and DFT-calculated γ-cleavage Gibbs activation energies was identified for the various substrates. In light of these data, we propose that the γ-cleavage step in the catalytic reaction pathway is rate-limiting. This conclusion has direct implications for the rational design of substrates or inhibitors aimed at regulating MGL activity.
Assuntos
Liases de Carbono-Enxofre/metabolismo , Metionina/metabolismo , Liases de Carbono-Enxofre/química , Catálise , Cisteína/metabolismo , Cinética , Metionina/análogos & derivados , Metionina/química , Porphyromonas gingivalis/metabolismo , Espectrofotometria/métodos , Especificidade por SubstratoRESUMO
The heme enzyme indoleamine 2,3-dioxygenase-1 (IDO1) catalyzes the first reaction of l-tryptophan oxidation along the kynurenine pathway. IDO1 is a central immunoregulatory enzyme with important implications for inflammation, infectious disease, autoimmune disorders, and cancer. Here we demonstrate that IDO1 is a mammalian nitrite reductase capable of chemically reducing nitrite to nitric oxide (NO) under hypoxia. Ultraviolet-visible absorption and resonance Raman spectroscopy showed that incubation of dithionite-reduced, ferrous-IDO1 protein (FeII-IDO1) with nitrite under anaerobic conditions resulted in the time-dependent formation of an FeII-nitrosyl IDO1 species, which was inhibited by substrate l-tryptophan, dependent on the concentration of nitrite or IDO1, and independent of the concentration of the reductant, dithionite. The bimolecular rate constant for IDO1 nitrite reductase activity was determined as 5.4 M-1 s-1 (pH 7.4, 23 °C), which was comparable to that measured for myoglobin (3.6 M-1 s-1; pH 7.4, 23 °C), an efficient and biologically important mammalian heme-based nitrite reductase. IDO1 nitrite reductase activity was pH-dependent but differed with myoglobin in that it showed a reduced proton dependency at pH >7. Electron paramagnetic resonance studies measuring NO production showed that the conventional IDO1 dioxygenase reducing cofactors, ascorbate and methylene blue, enhanced IDO1's nitrite reductase activity and the time- and IDO1 concentration-dependent release of NO in a manner inhibited by l-tryptophan or the IDO inhibitor 1-methyl-l-tryptophan. These data identify IDO1 as an efficient mammalian nitrite reductase that is capable of generating NO under anaerobic conditions. IDO1's nitrite reductase activity may have important implications for the enzyme's biological actions when expressed within hypoxic tissues.