Metal specificity is correlated with two crucial active site residues in Escherichia coli alkaline phosphatase.
Escherichia coli alkaline phosphatase exhibits maximal activity when Zn(2+) fills the M1 and M2 metal sites and Mg(2+) fills the M3 metal site. When other metals replace the zinc and magnesium, the catalytic efficiency is reduced by more than 5000-fold. Alkaline phosphatases from organisms such as Thermotoga maritima and Bacillus subtilis require cobalt for maximal activity and function poorly with zinc and magnesium. Previous studies have shown that the D153H alkaline phosphatase exhibited very little activity in the presence of cobalt, while the K328W and especially the D153H/K328W mutant enzymes can use cobalt for catalysis. To understand the structural basis for the altered metal specificity and the ability of the D153H/K328W enzyme to utilize cobalt for catalysis, we determined the structures of the inactive wild-type E. coli enzyme with cobalt (WT_Co) and the structure of the active D153H/K328W enzyme with cobalt (HW_Co). The structural data reveal differences in the metal coordination and in the strength of the interaction with the product phosphate (P(i)). Since release of P(i) is the slow step in the mechanism at alkaline pH, the enhanced binding of P(i) in the WT_Co structure explains the observed decrease in activity, while the weakened binding of P(i) in the HW_Co structure explains the observed increase in activity. These alterations in P(i) affinity are directly related to alterations in the coordination of the metals in the active site of the enzyme.
Wang
Jie
J
Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA.
Stieglitz
Kimberly A
KA
Kantrowitz
Evan R
ER
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008239:Lysine / Q000235:genetics; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008670:Metals / Q000737:chemistry* / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry; D014364:Tryptophan / Q000235:genetics; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D029968:Escherichia coli Proteins; D008024:Ligands; D008670:Metals; D010710:Phosphates; D001224:Aspartic Acid; D003035:Cobalt; D006639:Histidine; D014364:Tryptophan; D000469:Alkaline Phosphatase; D008274:Magnesium; D015032:Zinc; D008239:Lysine