2.3. Inhibition Experiments with Purified Recombinant SHMT Isoforms

Inhibition of SHMT activity by metformin was assessed using an established competitive binding assay [20,21]. The assay is based on the spectrophotometric measurement of the quinonoid intermediate that develops when both glycine and formyl-THF bind to SHMT, forming an enzyme/glycine/formyl-folate ternary complex [20]. The quinonoid intermediate, which has an intense absorption band with a maximum at ~500 nm, derives from the deprotonation of glycine, but it can accumulate to a measurable extent solely when a folate ligand is also bound to SHMT and a ternary complex is formed [22]. Accordingly, the absorbance at 500 nm is proportional to the fraction of enzyme present as a ternary complex. Metformin was dissolved in water to 50 mg/mL (0.3 mol/L).

Exploratory experiments were performed with 5 μmol/L solutions of purified human recombinant SHMT1 and SHMT2, which were incubated with a saturating concentration of glycine (3 mmol/L) in the presence of 5 μmol/L formyl-THF and a low concentration range of metformin (0–30 mmol/L) in 20 mmol/L potassium phosphate buffer, pH 7.2. All measurements were performed in triplicate. Inhibition curves were fitted to Equation (1), in which [I] is the concentration of metformin, to obtain the observed apparent inhibition constants (K_i).

In more complete assays with SHMT2 (5 μmol/L) enzyme solutions, when formyl-THF was the varying ligand (2.5–160 μmol/L), glycine was maintained at 10 mmol/L, and when glycine was the varying ligand (0.04–10 mmol/L) the formyl-THF concentration was maintained at 160 μmol/L. In both cases, the buffer used was 20 mmol/L potassium phosphate buffer (pH 7.8), and metformin concentrations varied between 0 and 200 mmol/L. In all assays, formyl-THF was added as the last component and, after rapid manual mixing, the absorbance change at 502 nm was recorded using a Hewlett-Packard 8453 diode-array spectrophotometer (Agilent Technologies, Santa Clara, CA, USA).

The data obtained from the two series of experiments were employed to generate saturation curves and double reciprocal plots, which were globally fitted to Equation (2).

in which v_o is the initial velocity, K_M is the Michaelis–Menten constant, V_MAX is the maximum velocity and