Purification and characterization of glyoxylate synthetase from greening potato-tuber chloroplasts.
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 mumol . mg protein-1 x 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 alpha-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 degrees 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.
Janave
M T
MT
Food Technology and Enzyme Engineering Division, Bhabha Atomic Research Centre, Bombay, India.
Ramaswamy
N K
NK
Nair
P M
PM
D000630:Aminopterin / Q000494:pharmacology; D002736:Chloroplasts / Q000201:enzymology* / Q000528:radiation effects; D004220:Disulfides / Q000032:analysis; D004789:Enzyme Activation; D005296:Ferrous Compounds / Q000494:pharmacology; D005561:Formates / Q000378:metabolism; D006038:Glyoxylates / Q000378:metabolism; D008027:Light; D008970:Molecular Weight; D007652:Oxo-Acid-Lyases / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D010618:Phenanthrolines / Q000494:pharmacology; D011198:Solanum tuberosum / Q000201:enzymology* / Q000528:radiation effects; D013050:Spectrometry, Fluorescence; D013763:Tetrahydrofolates / Q000494:pharmacology
D004220:Disulfides; D005296:Ferrous Compounds; D005561:Formates; D006038:Glyoxylates; D010618:Phenanthrolines; D013763:Tetrahydrofolates; C030544:formic acid; C020748:ferrous sulfate; C030371:5,6,7,8-tetrahydrofolic acid; D007652:Oxo-Acid-Lyases; C081991:glyoxylate synthetase; D000630:Aminopterin; C025205:1,10-phenanthroline