PURIFICATION AND CHARACTERIZATION OF GLYOXYLATE SYNTHETASE FROM GREENING POTATO-TUBER CHLOROPLASTS

Glyoxylate synthetase catalyzing the condensation of two formate molec ules 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 mumol . mg protein-1 . 5 min-1 was obt ained by this procedure. The reaction product was identified as glyoxy late. The enzyme was a tetramer having a molecular mass of 160 kDa wit h 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 aci d to the reaction mixture. The requirement for Fe2+ and tetrahydrofoli c acid was confirmed from the inhibition of enzyme by O-phenanthroline and a-aminopterin, respectively. The presence of a bound folate in th e enzyme was indicated by the fluorescence emission at 450 nm and turb idity development in a Lactobacillus casei growth test. Fluorescence e mission at 450 nm upon excitation at 280 nm indicated that the bound f olate and the aromatic amino-acid residues of the enzyme were in close vicinity. The enzyme was maximally active at 25-degrees-C and exhibit ed a pH optimum at 7.0. The concentration of substrate was optimal at 5.0 mM and K(m) for substrate was found to be 1.4 mM. Activation by Fe 2+ did not alter the K(m) but caused an increase in V(max). 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 t wo 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 .