|کد مقاله||کد نشریه||سال انتشار||مقاله انگلیسی||ترجمه فارسی||نسخه تمام متن|
|1172677||961527||2016||6 صفحه PDF||ندارد||دانلود رایگان|
• A new assay for MGL-catalyzed γ-cleavage of methionine and analogues is reported.
• The assay continuously monitors α-ketobutyrate production by absorbance at 315 nm.
• Km and kcat for γ-cleavage reactions by MGL from P. gingivalis are determined.
In this article, we present a new, easy-to-implement assay for methionine γ-lyase (MGL)-catalyzed γ-elimination reactions of l-methionine and its analogues that produce α-ketobutyrate (α-KB) as product. The assay employs ultraviolet–visible (UV–Vis) spectrophotometry to continuously monitor the rate of formation of α-KB by its absorbance at 315 nm. We also employ a nonlinear data analysis method that obviates the need for an “initial slope” determination, which can introduce errors when the progress curves are nonlinear. The spectrophotometric assay is validated through product analysis by 1H NMR (nuclear magnetic resonance), which showed that under the conditions of study l-methionine (l-met) and l-methionine sulfone (l-met sulfone) substrates were converted to α-KB product with greater than 99% yield. Using this assay method, we determined for the first time the Michaelis–Menten parameters for a recombinant form of MGL from Porphyromonas gingivalis, obtaining respective kcat and Km values of 328 ± 8 min−1 and 1.2 ± 0.1 mM for l-met γ-elimination and 2048 ± 59 min−1 and 38 ± 2 mM for l-met sulfone γ-elimination reactions. We envisage that this assay method will be useful for determining the activity of MGL γ-elimination reactions that produce α-KB as the end product.
Journal: Analytical Biochemistry - Volume 507, 15 August 2016, Pages 21–26