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.
Waditee
R
R
Laboratory of Biochemical Products, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
Incharoensakdi
A
A
D001622:Betaine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D050940:Betaine-Homocysteine S-Methyltransferase; D002794:Choline / Q000494:pharmacology; D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D000458:Cyanobacteria / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006710:Homocysteine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008970:Molecular Weight; D012521:Sarcosine / Q000031:analogs & derivatives / Q000494:pharmacology
D004791:Enzyme Inhibitors; D006710:Homocysteine; D001622:Betaine; C026820:betaine aldehyde; C025138:dimethylglycine; D008780:Methyltransferases; D050940:Betaine-Homocysteine S-Methyltransferase; D002794:Choline; D012521:Sarcosine