Studies on the catalytic mechanism of pig purple acid phosphatase.
Several independent experiments failed to reveal any evidence in support of the involvement of a phosphoryl-enzyme intermediate in the catalytic mechanism of pig allantoic fluid purple acid phosphatase: (i) attempts to label enzyme with phosphate derived from [32P]p-nitrophenyl phosphate were unsuccessful; (ii) values of kcat for a series of phosphate derivative varied over a wide range, with the enzyme showing a marked preference for activated ester and anhydride substrates over those with a stable leaving group; (iii) burst titrations revealed a "burst" of p-nitrophenol from p-nitrophenyl phosphate only when the enzyme was added after the substrate, suggesting that this result was an artifact of the order of addition of reagents; (iv) transphosphorylation from p-nitrophenyl phosphate to acceptor alcohols could not be detected, even under conditions where a transphosphorylation to hydrolysis ratio as low as 0.015 could have been measured; (v) enzyme-catalyzed exchange of 180 between phosphate and water was demonstrated, although at a rate much slower than that observed for other phosphatases where the involvement of a phosphoryl-enzyme intermediate in the mechanism has been clearly established. The present results are compared with those obtained in similar studies on other phosphatases, particularly the highly homologous beef spleen purple acid phosphatase, and their implications for the catalytic mechanism of the purple acid phosphatases are discussed.
Wynne
C J
CJ
Department of Biochemistry, University of Queensland, Australia.
Hamilton
S E
SE
Dionysius
D A
DA
Beck
J L
JL
de Jersey
J
J
D000135:Acid Phosphatase / Q000378:metabolism*; D000482:Allantois / Q000201:enzymology; D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D001826:Body Fluids / Q000201:enzymology; D002384:Catalysis; D000431:Ethanol / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010766:Phosphorylation; D013379:Substrate Specificity; D013552:Swine; D014270:Trifluoroethanol / Q000378:metabolism
D000814:Aniline Compounds; D006023:Glycoproteins; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D000431:Ethanol; C068798:4-aminophenylphosphate; D014270:Trifluoroethanol; C091137:purple acid phosphatase; D000135:Acid Phosphatase; D005990:Glycerol; D010100:Oxygen; C024836:4-nitrophenol