Purification and kinetic properties of betaine-homocysteine methyltransferase from Aphanothece halophytica.

Betaine-homocysteine methyl transferase (BHMT) from Aphanothece halophytica was purified to homogeneity by hydroxyapatite, DEAE-Sepharose CL-6B and Sephadex G-200 column chromatography. A 24-fold purification and 11% overall yield were achieved with a specific activity of 595 nmol h(-1) mg(-1). The subunit molecular weight was determined to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the native enzyme was found to have a molecular weight of 350 kDa, suggesting an octameric structure of the enzyme. The enzyme shows optimum activity at 37 degrees C, pH 7.5. The apparent Km values for glycinebetaine and L-homocysteine were 4.3 mM and 1.3 mM, respectively. The enzyme was 70% inactivated by 5 mM dimethylglycine whereas the same concentration of sarcosine slightly inactivated the enzyme. Two analogs of glycinebetaine were also tested for enzyme inactivation and it was found that 5 mM choline inactivated 60% of the enzyme activity and 2.5 mM betaine aldehyde completely abolished the enzyme activity. NaCl at 200 mM or higher also completely inactivated the enzyme.