Active-site-mutagenesis study of rat liver betaine-homocysteine S-methyltransferase.
A site-directed-mutagenesis study of putative active-site residues in rat liver betaine-homocysteine S-methyltransferase has been carried out. Identification of these amino acids was based on data derived from a structural model of the enzyme. No alterations in the CD spectra or the gel-filtration chromatography elution pattern were observed with the mutants, thus suggesting no modification in the secondary structure content or in the association state of the proteins. All the mutants obtained showed a reduction of the enzyme activity, the most dramatic effect being that of Glu(159), followed by Tyr(77) and Asp(26). Changes in affinity for either of the substrates, homocysteine or betaine, were detected when substitutions were performed of Glu(21), Asp(26), Phe(74) and Cys(186). Interestingly, Asp(26), postulated to be involved in homocysteine binding, has a strong effect on affinity for betaine. The relevance of these results is discussed in the light of very recent structural data obtained for the human enzyme.
González
Beatriz
B
Instituto de Química-Física Rocasolano (CSIC), Serrano 119, 28006 Madrid, Spain.
Campillo
Nuria
N
Garrido
Francisco
F
Gasset
María
M
Sanz-Aparicio
Juliana
J
Pajares
María A
MA
D000243:Adenosine Deaminase / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001622:Betaine / Q000378:metabolism; D050940:Betaine-Homocysteine S-Methyltransferase; D001665:Binding Sites; D002942:Circular Dichroism; D006710:Homocysteine / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment
D011993:Recombinant Fusion Proteins; D006710:Homocysteine; D001622:Betaine; D008780:Methyltransferases; C494548:BHMT protein, human; D050940:Betaine-Homocysteine S-Methyltransferase; C494549:Bhmt protein, mouse; C494550:Bhmt protein, rat; D000243:Adenosine Deaminase