Nicotinamide-adenine dinucleotide inhibition of pig kidney alkaline phosphatase.
1. The interaction of NAD+, NADH and various nucleotide analogues with pig kidney alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum) EC 3.1.3.1) has been investigated by kinetic means. Some inhibitors act uncompetitively whereas others markedly increase the slopes of double reciprocal plots suggesting they have some affinity for the free enzyme. 2. The compounds seem to bind to alkaline phosphatase through interactions of their bases with a relatively non-specific region of the enzyme, although it is likely that for those nucleotides having some affinity for the free enzyme there is some attraction between the pyrophosphate backbone and the active site. 3. From studies of the effect of NAD+ and NADH on ATPase activity it was concluded that the substrate inhibition that is characteristic of the ATPase activity of alkaline phosphatase originates from binding of ATP to the site assumed to exist for NAD+ and NADH. The potentiation of NAD+-inhibition of ATPase activity by Mg-2+ is probably a result of the depletion of [ATP-4-] the true substrate. The depletion allows NAD+ to complete more effectively for the active site. 4. Binding of NADH is favoured by protonation of an enzymic group with a pK of approx. 9.0 belonging possibly to a tyrosine residue or a zinc hydrate. 5. A large entropy decrease was found to accompany the binding of NAD+ and NADH to alkaline phosphatase. This may be further evidence of an "induced-fit" mechanism previously suspected because of the synergistic inhibitory effects of adenosine and nicotinamide.
D000251:Adenosine Triphosphatases / Q000032:analysis; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors*; D000818:Animals; D001665:Binding Sites; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009243:NAD / Q000494:pharmacology*; D009536:Niacinamide / Q000494:pharmacology; D011485:Protein Binding; D013552:Swine; D013696:Temperature; D013774:Tetranitromethane / Q000494:pharmacology; D013816:Thermodynamics
D009243:NAD; D009536:Niacinamide; D000255:Adenosine Triphosphate; D000469:Alkaline Phosphatase; D000251:Adenosine Triphosphatases; D008274:Magnesium; D013774:Tetranitromethane