A coupled photometric assay for characterization of S-adenosyl-l-homocysteine hydrolases in the physiological hydrolytic direction

• Published in: New biotechnology 2017-10, Vol.39 (Pt A), p.11-17
• Main Authors: Kailing, Lyn L., Bertinetti, Daniela, Herberg, Friedrich W., Pavlidis, Ioannis V.
• Format: Article
• Language: English
• Subjects: Adenosine; Adenosine diphosphate; Adenosine kinase; Adenosine Monophosphate - pharmacology; Adenosylhomocysteinase - isolation & purification; Adenosylhomocysteinase - metabolism; AMP; Assaying; Biocatalysis; Biocatalysts; Bradyrhizobium - enzymology; Chemical reactions; Enzymes; Equilibrium shift; Homocysteine; Homocysteine - pharmacology; Hydrogen-Ion Concentration; Hydrolase; Hydrolysis; Kinetic assay; Kinetics; L-Homocysteine; L-Lactate dehydrogenase; Lactate dehydrogenase; Lactic acid; Methionine; Methylation; NADH; Nicotinamide adenine dinucleotide; Photometry; Photometry - methods; Pyruvate kinase; Pyruvic acid; Regeneration; S-Adenosyl-l-homocysteine; S-Adenosylhomocysteine - chemistry; S-Adenosylhomocysteine - metabolism; Temperature
• ISSN: 1871-6784; 1876-4347
• DOI: 10.1016/j.nbt.2017.04.005

[Display omitted] •Development of a real-time assay for S-adenosyl-L-homocysteine (SAH) hydrolases.•The assay is fast, specific and not disturbed by the ubiquitous cofactor NAD+.•Simple instrumentation: the assay requires only a standard photometer.•Showcase: kinetic characterization of an SAH hydrolase from Bradyrhizobium elkanii. S-Adenosyl-l-homocysteine hydrolases (SAHases) are important metabolic enzymes and their dysregulation is associated with some severe diseases. In vivo they catalyze the hydrolysis of S-adenosyl-l-homocysteine (SAH), the by-product of methylation reactions in various organisms. SAH is a potent inhibitor of methyltransferases, thus its removal from the equilibrium is an important requirement for methylation reactions. SAH hydrolysis is also the first step in the cellular regeneration process of the methyl donor S-adenosyl-l-methionine (SAM). However, in vitro the equilibrium lies towards the synthetic direction. To enable characterization of SAHases in the physiologically relevant direction, we have developed a coupled photometric assay that shifts the equilibrium towards hydrolysis by removing the product adenosine, using a high affinity adenosine kinase (AK). This converts adenosine to AMP and thereby forms equimolar amounts of ADP, which is phosphorylated by a pyruvate kinase (PK), in turn releasing pyruvate. The readout of the assay is the consumption of NADH during the lactate dehydrogenase (LDH) catalyzed reduction of pyruvate to lactic acid. The applicability of the assay is showcased for the determination of the kinetic constants of an SAHase from Bradyrhizobium elkanii (KM,SAH 41±5μM, vmax,SAH 25±1μM/min with 0.13mg/mL enzyme). This assay is a valuable tool for in vitro characterization of SAHases with biotechnological potential, and for monitoring SAHase activity in diagnostics.