Glutamic acid residues as metal ligands in the active site of Escherichia coli alkaline phosphatase.
Four independent mutations were introduced to the Escherichia coli alkaline phosphatase active site, and the resulting enzymes characterized to study the effects of Glu as a metal ligand. The mutations D51E and D153E were created to study the effects of lengthening the carboxyl group by one methylene unit at the metal interaction site. The D51E enzyme had drastically reduced activity and lost one zinc per active site, demonstrating importance of the position of Asp(51). The D153E enzyme had an increased k(cat) in the presence of high concentrations of Mg(2+), along with a decreased Mg(2+) affinity as compared to the wild-type enzyme. The H331E and H412E enzymes were created to probe the requirement for a nitrogen-containing metal ligand at the Zn(1) site. The H331E enzyme had greatly decreased activity, and lost one zinc per active site. In the absence of high concentrations of Zn(2+), dephosphorylation occurs at an extremely reduced rate for the H412E enzyme, and like the H331E enzyme, metal affinity is reduced. Except at the 153 position, Glu is not an acceptable metal chelating amino acid at these positions in the E. coli alkaline phosphatase active site.
Wojciechowski
Cheryl L
CL
Department of Chemistry, Boston College, Merkert Chemistry Center, 140 Commonwealth Avenue, Chestnut Hill, MA 02467, USA.
Kantrowitz
Evan R
ER
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D013329:Structure-Activity Relationship; D015032:Zinc / Q000378:metabolism
D029968:Escherichia coli Proteins; D008024:Ligands; D008670:Metals; D001224:Aspartic Acid; D018698:Glutamic Acid; D006639:Histidine; D000469:Alkaline Phosphatase; D008274:Magnesium; D015032:Zinc