title: A continuous spectrophotometric assay and nonlinear kinetic analysis of methionine γ-lyase catalysis.
abstract: 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 (1)H 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.
authors.affiliation: Department of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton, FL 33431, USA.
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Foo
authors.affiliation: Department of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton, FL 33431, USA. Electronic address: terentis@fau.edu.
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Terentis
authors.affiliation: Department of Biochemistry, College of Medical Sciences, Nova Southeastern University, Fort Lauderdale, FL 33328, USA. Electronic address: venk@nova.edu.
authors.forename: Kallidaikurichi V
authors.initials: KV
authors.lastname: Venkatachalam
mesh_terms: D055162:Biocatalysis; D002087:Butyrates / Q000737:chemistry / Q000378:metabolism; D013437:Carbon-Sulfur Lyases / Q000032:analysis* / Q000378:metabolism; D007700:Kinetics; D008715:Methionine / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D016966:Porphyromonas gingivalis / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet
chemical_list: D002087:Butyrates; C005087:alpha-ketobutyric acid; D008715:Methionine; D013437:Carbon-Sulfur Lyases; C021558:L-methionine gamma-lyase
keywords: Enzyme catalysis; Methionine γ-lyase; Porphyromonas gingivalis; Pyridoxal-5′-phosphate; UV–Vis spectrophotometry