HUMAN BETAINE-HOMOCYSTEINE METHYLTRANSFERASE IS A ZINC METALLOENZYME

We have overexpressed recombinant human liver betaine-homocysteine met hyltransferase (BHMT; EC 2.1.1.5) in Escherichia coli and have purifie d the enzyme to homogeneity, The Michaelis constants for betaine and L -homocysteine are 2.2 mM and 4 mu M, respectively. Analysis of the pur e protein for metals by inductively coupled plasma emission spectromet ry indicate that the recombinant enzyme contains zinc, Extensive dialy sis in buffer containing high levels of EDTA could not strip the prote in of zinc. However, dialysis against buffer containing EDTA and methy l methanethiosulfonate, followed by buffer containing EDTA and dithiot hreitol, could remove zinc from the enzyme with concomitant loss of ac tivity. Dialyzing the zinc-depleted enzyme against buffer containing 1 M urea and 2 mM zinc, followed by dialysis with buffer alone, complet ely restored BHMT activity and zinc content. BHMT was also partially p urified from human liver. The purest BHMT-containing fractions also co ntained zinc and the enzyme was kinetically indistinguishable from the recombinant enzyme. As with the recombinant enzyme, the partially pur ified 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 B HMT is a major zinc metalloenzyme in liver and that cysteine residues are likely involved in zinc binding. (C) 1998 Academic Press.