Structural requirements for catalysis and dimerization of human methionine adenosyltransferase I/III.
We have used site-directed mutagenesis to probe the structural requirements for catalysis and dimerization of human hepatic methionine adenosyltransferase (hMAT). We built a homology model of the dimeric hMAT III inferred by the crystal structure of the highly homologous Escherichia coli MAT dimer. The active sites of both enzymes comprise the same amino acids and are located in the inter-subunit interface. All of the amino acids predicted to be in the hMAT III active site were mutated, as well as residues in a conserved ATP binding region. All of the mutations except one severely affected catalytic activity. On the other hand, dimerization was affected only by single mutations of three different residues, all on one monomer. The homology model suggested that the side chains of these residues stabilized the monomer and participated in a bridge between subunits consisting of a network of metal and phosphate ions. In agreement with this observation, we demonstrated that dimerization cannot occur in the absence of phosphate.
Chamberlin
M E
ME
Heritable Disorders Branch, National Institute of Child Health and Human Development, Bethesda, Maryland, 20892, USA.
Ubagai
T
T
Pao
V Y
VY
Pearlstein
R A
RA
Yang Chou
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D003062:Codon / Q000235:genetics; D017124:Conserved Sequence; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008716:Methionine Adenosyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000737:chemistry; D020836:Protein Structure, Quaternary; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D003062:Codon; D007527:Isoenzymes; D010710:Phosphates; D008716:Methionine Adenosyltransferase