Dissecting the catalytic mechanism of betaine-homocysteine S-methyltransferase by use of intrinsic tryptophan fluorescence and site-directed mutagenesis.
Betaine-homocysteine S-methyltransferase (BHMT) is a zinc-dependent enzyme that catalyzes the transfer of a methyl group from glycine betaine (Bet) to homocysteine (Hcy) to form dimethylglycine (DMG) and methionine (Met). Previous studies in other laboratories have indicated that catalysis proceeds through the formation of a ternary complex, with a transition state mimicked by the inhibitor S-(delta-carboxybutyl)-l-homocysteine (CBHcy). Using changes in intrinsic tryptophan fluorescence to determine the affinity of human BHMT for substrates, products, or CBHcy, we now demonstrate that the enzyme-substrate complex reaches its transition state through an ordered bi-bi mechanism in which Hcy is the first substrate to bind and Met is the last product released. Hcy, Met, and CBHcy bind to the enzyme to form binary complexes with K(d) values of 7.9, 6.9, and 0.28 microM, respectively. Binary complexes with Bet and DMG cannot be detected with fluorescence as a probe, but Bet and DMG bind tightly to BHMT-Hcy to form ternary complexes with K(d) values of 1.1 and 0.73 microM, respectively. Mutation of each of the seven tryptophan residues in human BHMT provides evidence that the enzyme undergoes two distinct conformational changes that are reflected in the fluorescence of the enzyme. The first is induced when Hcy binds, and the second, when Bet binds. As predicted by the crystal structure of BHMT, the amino acids Trp44 and Tyr160 are involved in binding Bet, and Glu159 in binding Hcy. Replacing these residues by site-directed mutagenesis significantly reduces the catalytic efficiency (V(max)/K(m)) of the enzyme. Replacing Tyr77 with Phe abolishes enzyme activity.
Castro
Carmen
C
Department of Food Science and Human Nutrition, University of Illinois, Urbana, Illinois 61801, USA.
Gratson
Alejandra A
AA
Evans
John C
JC
Jiracek
Jiri
J
Collinsová
Michaela
M
Ludwig
Martha L
ML
Garrow
Timothy A
TA
D001622:Betaine / Q000378:metabolism; D050940:Betaine-Homocysteine S-Methyltransferase; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D004791:Enzyme Inhibitors / Q000737:chemistry; D018698:Glutamic Acid / Q000235:genetics; D006710:Homocysteine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008715:Methionine / Q000378:metabolism; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D012521:Sarcosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity / Q000235:genetics; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics; D014443:Tyrosine / Q000235:genetics
D004791:Enzyme Inhibitors; D008024:Ligands; D006710:Homocysteine; D018698:Glutamic Acid; D001622:Betaine; D014443:Tyrosine; C025138:dimethylglycine; C039751:5-(delta-carboxybutyl)homocysteine; D014364:Tryptophan; D008715:Methionine; D008780:Methyltransferases; C494548:BHMT protein, human; D050940:Betaine-Homocysteine S-Methyltransferase; D012521:Sarcosine