A histidine thiol 100 kDa, tetrameric acid phosphatase from lentil, Lens esculenta, seeds with the characteristics of protein tyrosine phosphatases.
A non-specific acid phosphatase (APase) hydrolysing L-tyrosine-O-phosphate and 3'-AMP was purified to electrophoretic homogeneity from mature lentil seeds with apparent native molecular mass of 100 kDa and subunit molecular mass of 24 kDa. These activities appear to reside on the same protein which shows a single band in native and SDS-PAGE. The pH optimum is 5.5, while the K(m) (mM) and V(max) (micromoles/min/mg protein) for p-nitrophenyl phosphate (pNPP) are 0.7 and 9.2 and for L-tyrosine-O-phosphate 1.4 and 10.1, respectively, at 30 degrees C and for 3'-AMP, 2 and 4.4 at 37 degrees C. The protein also hydrolyses other phosphomonoesters to a lesser extent. L-Tyrosine-O-phosphate, 3'-AMP and pNPP hydrolysis is potently inhibited by micromolar orthovanadate and also to nearly the same extent by sodium fluoride, potassium tartrate and metal ions. Histidine and cysteine are likely to be involved in the catalysis. Thermal inactivation studies indicate that the active site conformations for pNPP and 3'-AMP hydrolytic activities are different. The enzyme shows the characteristics of the animal protein tyrosine phosphatase.
Roknabadi
S M
SM
Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi-221 005, India.
Bose
S K
SK
Taneja
V
V
D000249:Adenosine Monophosphate / Q000378:metabolism; D000814:Aniline Compounds / Q000378:metabolism; D001665:Binding Sites; D002853:Chromatography, Liquid / Q000379:methods; D003545:Cysteine / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007887:Fabaceae / Q000201:enzymology*; D006639:Histidine / Q000032:analysis; D007700:Kinetics; D008970:Molecular Weight; D009943:Organophosphorus Compounds / Q000378:metabolism; D010946:Plants, Medicinal; D011487:Protein Conformation; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000201:enzymology; D013379:Substrate Specificity
D000814:Aniline Compounds; D004791:Enzyme Inhibitors; D009943:Organophosphorus Compounds; D000249:Adenosine Monophosphate; D006639:Histidine; C068798:4-aminophenylphosphate; D017027:Protein Tyrosine Phosphatases; D003545:Cysteine