A continuous spectrophotometric assay and nonlinear kinetic analysis of methionine gamma-lyase catalysis

作者：Foo Timothy C; Terentis Andrew C; Venkatachalam Kallidaikurichi V

来源：Analytical Biochemistry, 2016, 507: 21-26.

DOI：10.1016/j.ab.2016.05.010

• Methionine gamma-lyase
• Pyridoxal-5 '-phosphate
• Porphyromonas gingivalis
• Enzyme catalysis
• UV-Vis spectrophotometry

In this article, we present a new, easy-to-implement assay for methionine gamma-lyase (MGL)-catalyzed gamma-elimination reactions of L-methionine and its analogues that produce alpha-ketobutyrate (alpha-KB) as product. The assay employs ultraviolet-visible (UV-Vis) spectrophotometry to continuously monitor the rate of formation of alpha-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 H-1 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 alpha-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 k(cat) and K-m values of 328 +/- 8 min(-1) and 1.2 +/- 0.1 mM for L-met gamma-elimination and 2048 +/- 59 min(-1) and 38 +/- 2 mM for L-met sulfone gamma-elimination reactions. We envisage that this assay method will be useful for determining the activity of MGL gamma-elimination reactions that produce alpha-KB as the end product.

• 出版日期2016-8-15
• 单位东南大学