Biochemical characterization of the class B acid phosphatase (AphA) of Escherichia coli MG1655.
The AphA enzyme of Escherichia coli, a molecular class B periplasmic phosphatase that belongs to the DDDD superfamily of phosphohydrolases, was purified and subjected to biochemical characterization. Kinetic analysis with several substrates revealed that the enzyme essentially behaves as a broad-spectrum nucleotidase highly active on 3'- and 5'-mononucleotides and monodeoxynucleotides, but not active on cyclic nucleotides, or nucleotides di- and triphosphate. Mononucleotides are degraded to nucleosides, and AphA apparently does not exhibit any nucleotide phosphomutase activity. However, it can transphosphorylate nucleosides in the presence of phosphate donors. Kinetic properties of AphA are consistent with structural data, and suggest a role for the hydrophobic pocket present in the active site crevice, made by residues Phe 56, Leu71, Trp77 and Tyr193, in conferring preferential substrate specificity by accommodating compounds with aromatic rings. AphA was inhibited by several chelating agents, including EDTA, EGTA, 1,10-phenanthroline and dipicolinic acid, with EDTA being apparently the most powerful inhibitor.
Passariello
Claudio
C
Dipartimento di Scienze di Sanità Pubblica, Università di Roma La Sapienza, 00185 Rome, Italy.
Forleo
Costantino
C
Micheli
Vanna
V
Schippa
Serena
S
Leone
Rosalida
R
Mangani
Stefano
S
Thaller
Maria Cristina
MC
Rossolini
Gian Maria
GM
D000135:Acid Phosphatase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D002614:Chelating Agents / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D009711:Nucleotides / Q000378:metabolism; D020836:Protein Structure, Quaternary; D013379:Substrate Specificity; D013816:Thermodynamics
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D009711:Nucleotides; D000135:Acid Phosphatase