Substrate specificity of IphP, a cyanobacterial dual-specificity protein phosphatase with MAP kinase phosphatase activity.
The substrate specificity of the cyanobacterial dual-specificity protein phosphatase, IphP, was explored using a variety of potential substrates. The enzyme displayed phosphomonoesterase activity toward a broad range of peptide, protein, and low molecular weight organophosphate compounds. It displayed little or no hydrolase activity toward phosphodiesters, phosphoramides, carboxyl esters, or sulfoesters. However, it did display measurable pyrophosphatase activity, especially toward ADP and ATP. Among the low molecular weight phosphomonoesters, the presence of an aromatic ring either as part of the leaving group alcohol or immediately adjacent thereto, as in 5'-AMP, was a strong positive determinant for hydrolysis. Among peptide and protein substrates, a rough, but imperfect, correlation between charge character and hydrolysis was noted in which proteins and phosphorylation sites of an acidic nature seemed favored. Heparin affected IphP activity in a substrate-dependent manner. Toward small organophosphates, heparin had no significant effect, but it was inhibitory toward most protein and peptide substrates. However, toward phosphoseryl casein and MAP kinase, it enhanced activity as much as 10-fold. This enhancement was attributed to the ability of heparin to bind to these substrate proteins, as well as IphP, and recruit them to the same microenvironment.
Howell
L D
LD
Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg 24061-0308, USA.
Griffiths
C
C
Slade
L W
LW
Potts
M
M
Kennelly
P J
PJ
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000302:isolation & purification / Q000378:metabolism; D000458:Cyanobacteria / Q000201:enzymology*; D004926:Escherichia coli; D007700:Kinetics; D008969:Molecular Sequence Data; D010755:Organophosphates / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D010755:Organophosphates; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D017027:Protein Tyrosine Phosphatases