Rate-determining step of Escherichia coli alkaline phosphatase altered by the removal of a positive charge at the active center.
Escherichia coli alkaline phosphatase catalyzes both the nonspecific hydrolysis of phosphomonoesters and a transphosphorylation reaction in which phosphate is transferred to an alcohol via a phosphoseryl intermediate. The rate-determining step for the wild-type enzyme is pH dependent. At alkaline pH, release of the product phosphate from the noncovalent enzyme-phosphate complex determines the reaction rate, whereas at acidic pH hydrolysis of the covalent enzyme-phosphate complex controls the reaction rate. When the lysine at position 328 was substituted with a cysteine (K328C), the rate-determining step at pH 8.0 of the mutant enzyme was altered so that hydrolysis of the covalent intermediate became limiting rather than phosphate release. The transphosphorylation activity of the K328C enzyme was selectively enhanced, while the hydrolysis activity was reduced compared to that of the wild-type enzyme. The ratio of the transphosphorylation to the hydrolysis activities increased 28-fold for the K328C enzyme in comparison with the wild-type enzyme. Several other mutant enzymes for which a positive charge at the active center is removed by site-specific mutagenesis share this characteristic of the K328C enzyme. These results suggest that the positive charge at position 328 is at least partially responsible for maintaining the balance between the hydrolysis and transphosphorylation activities and plays an important role in determining the rate-limiting step of E. coli alkaline phosphatase.
Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167, USA.
L
L
Sun
D C
DC
Martin
E R
ER
Kantrowitz
D000409:Alanine / Q000235:genetics; D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000235:genetics; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000737:chemistry
D010710:Phosphates; D006639:Histidine; D000469:Alkaline Phosphatase; D008274:Magnesium; D008239:Lysine; D003545:Cysteine; D000409:Alanine