A continuous spectrophotometric assay and nonlinear kinetic analysis of methionine g-lyase catalysis Timothy C. Foo a , Andrew C. Terentis a, * , Kallidaikurichi V. Venkatachalam b, ** a Department of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton, FL 33431, USA b Department of Biochemistry, College of Medical Sciences, Nova Southeastern University, Fort Lauderdale, FL 33328, USA article info Article history: Received 27 January 2016 Received in revised form 8 April 2016 Accepted 13 May 2016 Available online 24 May 2016 Keywords: Methionine g-lyase Pyridoxal-5 0 -phosphate Porphyromonas gingivalis Enzyme catalysis UVeVis spectrophotometry abstract In this article, we present a new, easy-to-implement assay for methionine g-lyase (MGL)-catalyzed g- elimination reactions of L-methionine and its analogues that produce a-ketobutyrate (a-KB) as product. The assay employs ultravioletevisible (UVeVis) spectrophotometry to continuously monitor the rate of formation of a-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 a-KB product with greater than 99% yield. Using this assay method, we determined for the first time the MichaeliseMenten 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 g-elimination and 2048 ± 59 min 1 and 38 ± 2 mM for L-met sulfone g-elimination reactions. We envisage that this assay method will be useful for determining the activity of MGL g-elimination reactions that produce a-KB as the end product. © 2016 Elsevier Inc. All rights reserved. The pyridoxal 5 0 -phosphate (PLP) dependent enzyme, methio- nine g-lyase (MGL; EC 4.4.1.11), catalyzes the g-elimination of L- methionine (L-met) forming a-ketobutyrate (a-KB), ammonium, and methanethiol (Scheme 1). MGL also catalyzes g-replacement reactions of L-met with thiol compounds to form S-substituted L- met analogues as well as a-b-elimination and b-replacement of L- cysteine and various S-substituted L-cysteine analogues [1,2]. MGL is a globular protein that functions as a homotetramer, with each identical subunit (~43 kDa) containing a PLP cofactor [3]. MGL has been isolated from various sources such as pathogenic bacteria (e.g., Clostridium tetani [4], Porphyromonas gingivalis [5]) and pathogenic protozoa (e.g., Entamoeba histolytica [6], Trichomonas vaginalis [7]). Extensive in vitro biochemical and biophysical studies have been published on three recombinant MGL isoforms from Pseudomonas putida (¼ Pseudomonas ovalis) [8e10], Citrobacter freundii [3,11,12], and T. vaginalis [13,14]. Besides its physiological roles in organisms, MGL is also a therapeutic target for pathogenic diseases such as trichomoniasis, gingivitis, amoebiasis, and cancer [15]. An MGL substrate analogue, trifluoromethionine (TFM), is a prodrug that has emerged as a lead compound for a novel class of potent antibiotics [16,17]. Cancerous cells have a higher requirement for L-met; thus, exogenous MGL was used to reduce plasma L-met concentrations to inhibit tumor growth in several animal models [18e22]. Because bacterial- derived MGLs have a short half-life in serum, a human cys- tathionine-g-lyase mutant was recently engineered to exhibit MGL activity with a longer serum half-life [23]. In addition, cancer cells transfected with the P. gingivalis gene exhibited severe cell aggre- gation and death [24]. Thus, there are broad prospects for MGL's applications in medicine. However, there still lacks a thorough understanding of the mechanisms of the reactions catalyzed by MGL, which can be obtained from detailed enzyme kinetic studies. Such studies require a reliable and simple enzyme assay system and method of data analysis, both of which have been lacking for MGL. Most of the MGL enzymatic activity assays employed to date have involved derivatizing the product to a chromophoric species, Abbreviations used: PLP, pyridoxal 5 0 -phosphate; MGL, methionine g-lyase; L- met, L-methionine; a-KB, a-ketobutyrate; TFM, trifluoromethionine; MBTH, 3- methyl-2-benzothiazolinone hydrazone hydrochloride; DNPH, dinitrophenylhy- drazine; LDH, lactate dehydrogenase; L-met sulfone, L-methionine sulfone; UVeVis, ultravioletevisible; NMR, nuclear magnetic resonance; DTT, dithiothreitol. * Corresponding author. ** Corresponding author. E-mail addresses: terentis@fau.edu (A.C. Terentis), venk@nova.edu (K.V. Venkatachalam). Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio http://dx.doi.org/10.1016/j.ab.2016.05.010 0003-2697/© 2016 Elsevier Inc. All rights reserved. Analytical Biochemistry 507 (2016) 21e26