Purification and characterization of glyoxylate synthetase from greening potato‐tuber chloroplasts

Abstract

Glyoxylate synthetase catalyzing the condensation of two formate molecules into glyoxylate was purified to homogeneity by AcA‐34, Sepharose CL‐6B and DEAE‐Sepharose CL‐6B chromatography. A 150‐fold purification with a specific activity of 25 μmol · mg protein−1· 5 min−1 was obtained by this procedure. The reaction product was identified as glyoxylate. The enzyme was a tetramer having a molecular mass of 160 kDa with a subunit molecular mass of 40 kDa. The enzyme could be activated 3–4‐fold by the addition of 0.3 mM Fe2+ and 0.4 mM tetrahydrofolic acid to the reaction mixture. The requirement for Fe2+ and tetrahydrofolic acid was confirmed from the inhibition of enzyme by O‐phenanthroline and α‐aminopterin, respectively. The presence of a bound folate in the enzyme was indicated by the fluorescence emission at 450 nm and turbidity development in a Lactobacillus casei growth test. Fluorescence emission at 450 nm upon excitation at 280 nm indicated that the bound folate and the aromatic amino‐acid residues of the enzyme were in close vicinity. The enzyme was maximally active at 25°C and exhibited a pH optimum at 7.0. The concentration of substrate was optimal at 5.0 mM and Km for substrate was found to be 1.4 mM. Activation by Fe2+ did not alter the Km but caused an increase in Vmax. The enzyme contained about 14–16 disulfide linkages, of which two were found to be reduced by treatment with 2‐mercaptoethanol. The presence of excess 2‐mercaptoethanol in the enzyme was inhibitory, indicating that the two disulfide linkages reduced by 2‐mercaptoethanol were essential for activity. This was also confirmed by the inhibition of enzyme activity when reduced enzyme was treated with O‐phthalaldehyde, which formed a thioisoindole derivative with reduced thiol groups at the active site. Copyright © 1993, Wiley Blackwell. All rights reserved