Human betaine-homocysteine methyltransferase is a zinc metalloenzyme.
We have overexpressed recombinant human liver betaine-homocysteine methyltransferase (BHMT; EC 2.1.1.5) in Escherichia coli and have purified the enzyme to homogeneity. The Michaelis constants for betaine and l-homocysteine are 2.2 mM and 4 microM, respectively. Analysis of the pure protein for metals by inductively coupled plasma emission spectrometry indicate that the recombinant enzyme contains zinc. Extensive dialysis in buffer containing high levels of EDTA could not strip the protein of zinc. However, dialysis against buffer containing EDTA and methyl methanethiosulfonate, followed by buffer containing EDTA and dithiothreitol, could remove zinc from the enzyme with concomitant loss of activity. Dialyzing the zinc-depleted enzyme against buffer containing 1 M urea and 2 mM zinc, followed by dialysis with buffer alone, completely restored BHMT activity and zinc content. BHMT was also partially purified from human liver. The purest BHMT-containing fractions also contained zinc and the enzyme was kinetically indistinguishable from the recombinant enzyme. As with the recombinant enzyme, the partially purified human liver enzyme could be inactivated by treatment with methyl methanethiosulfonate, EDTA, and dithiothreitol. Reconstitution of the zinc-depleted enzyme completely restored activity. We conclude that BHMT is a major zinc metalloenzyme in liver and that cysteineresidues are likely involved in zinc binding.
Millian
N S
NS
Division of Nutritional Sciences, University of Illinois, Urbana, Illinois, 61801, USA.
Garrow
T A
TA
D050940:Betaine-Homocysteine S-Methyltransferase; D002384:Catalysis; D002848:Chromatography, DEAE-Cellulose; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008667:Metalloproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008780:Methyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015032:Zinc / Q000378:metabolism*
D008667:Metalloproteins; D011994:Recombinant Proteins; D008780:Methyltransferases; C494548:BHMT protein, human; D050940:Betaine-Homocysteine S-Methyltransferase; D015032:Zinc