Gamma cleavage is a rate-determining step in the gamma-elimination reaction of L-methionine analogues catalyzed by methionine-gamma-lyase
| Autor: | Andrew C. Terentis, Timothy C Foo, Kallidaikurichi V Venkatachalam, Jesse E Meacham |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Methionine gamma-lyase Stereochemistry Biophysics Biochemistry Catalysis Substrate Specificity Analytical Chemistry Sulfone 03 medical and health sciences Elimination reaction chemistry.chemical_compound Methionine 0302 clinical medicine Cysteine Enzyme kinetics Molecular Biology Substrate (chemistry) Rate-determining step Turnover number Carbon-Sulfur Lyases Kinetics 030104 developmental biology chemistry Spectrophotometry 030220 oncology & carcinogenesis Porphyromonas gingivalis |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1869:140652 |
| ISSN: | 1570-9639 |
| DOI: | 10.1016/j.bbapap.2021.140652 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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