Kinetic parameters for the cleaved substrate, and enzyme and substrate stability, vary with the phosphoacceptor in alkaline phosphatase catalysis.
Nine different isoenzymes and (or) isoforms of alkaline phosphatase (ALP; EC 3.1.3.1) from human tissue were studied with respect to Km and Vmax values for p-nitrophenyl phosphate (p-NPP) in seven different potential phosphoacceptors/buffers. Generally, the phosphoacceptors/buffers with the lowest affinity for p-NPP (highest Km values) gave the highest Vmax values; for the nine enzyme forms in this study, the mean Km and Vmax values were greatest in 2-(ethylamino) (EAE). The two amino-propanol buffers gave the lowest Km and Vmax values. The phosphoacceptors/buffers N-methyl-D-glucamine (MEG), diethanolamine, and Tris had intermediate Km and Vmax values. Hydrophilic liver ALP retained > 90% of its activity after 24 h at 30 degrees C in both 1.0 and 0.3 mol/L Tris and 2-amino-2-methyl-1,3-propanediol and in 0.3 mol/L MEG. This isoenzyme showed greatest inactivation upon prolonged exposure to 1.0 and 0.3 mol/L EAE, the activity at 24 h being approximately 50-66% of that at zero time. p-NPP underwent the greatest spontaneous degradation, approximately 2.5 times that of baseline levels, in 1 mol/L MEG. There was little degradation in all of the buffers tested at 0.3 mol/L or in Tris, EAE, and 2-amino-2-methyl-1-propanol at 1.0 mol/L.
Stinson
R A
RA
Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Canada.
D000469:Alkaline Phosphatase / Q000378:metabolism*; D002021:Buffers; D002384:Catalysis; D004795:Enzyme Stability; D019856:Ethanolamine; D004983:Ethanolamines / Q000378:metabolism; D005260:Female; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008536:Meglumine / Q000378:metabolism; D009596:Nitrophenols / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000378:metabolism*; D010920:Placenta / Q000201:enzymology; D011412:Propanolamines / Q000378:metabolism
D002021:Buffers; D004983:Ethanolamines; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D011412:Propanolamines; C008644:nitrophenylphosphate; D019856:Ethanolamine; D008536:Meglumine; C020283:diethanolamine; D000469:Alkaline Phosphatase; C006551:2-amino-2-methyl-1-propanol