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-GB and Se phadex 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 sulf atepolyacrylamide gel electrophoresis (SDS-PAGE), and the native enzyme was found to have a molecular weight of 350 kDa, suggesting an octameric struc ture of the enzyme. The enzyme shows optimum activity at 37 degreesC, 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 dimethylg lycine whereas the same concentration of sarcosine slightly inactivated the enzyme. Two analogs of glycinebetaine were also tested for enzyme inactiva tion and it was found that 5 mM choline inactivated 60% of the enzyme activ ity and 2.5 mM betaine aldehyde completely abolished the enzyme activity. N aCl at 200 mM or higher also completely inactivated the enzyme.