The inhibition of pig kidney alkaline phosphatase by oxidized or reduced nicotinamide-adenine dinucleotide and related compounds.
1. The inhibition of alkaline phosphatase by NAD(+), NADH, adenosine and nicotinamide was studied. 2. All of these substances except NAD(+) act as uncompetitive inhibitors, i.e. double-reciprocal plots are parallel. NAD(+), however, is a ;mixed' inhibitor of alkaline phosphatase and is less potent than NADH. 3. Inhibition studies with pairs of the inhibitors suggest that, in spite of the difference in type of inhibition, NAD(+) and NADH bind to alkaline phosphatase at a common site. Adenosine and nicotinamide also seem to bind at the NAD site and the binding of adenosine is facilitated by nicotinamide, and vice versa. 4. The facilitation may indicate the occurrence of an induced fit for NAD(+) and NADH. Attempts to desensitize alkaline phosphatase to NAD(+) and NADH inhibition by partial denaturation were unsuccessful. 5. The results are discussed in terms of a two-site model in which separate, but interacting, regions exist on the enzyme to accommodate the adenosine and nicotinamide moieties of NAD, and a single-site model in which the adenosine part of the molecule is bound preferentially and this interacts with the nicotinamide fraction. 6. The activity of alkaline phosphatase can be changed fourfold by alteration of the NAD(+)/NADH ratio. This sensitivity to the redox state of the coenzyme could be a means of controlling phosphatase activity.
D000241:Adenosine / Q000494:pharmacology; D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors*; D000818:Animals; D001665:Binding Sites; D004355:Drug Stability; D006146:Guanidines / Q000494:pharmacology; D007668:Kidney / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D008433:Mathematics; D008956:Models, Chemical; D009243:NAD / Q000494:pharmacology*; D009536:Niacinamide / Q000494:pharmacology; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D013053:Spectrophotometry; D013552:Swine; D013997:Time Factors
D006146:Guanidines; D009243:NAD; D009536:Niacinamide; D000469:Alkaline Phosphatase; D000241:Adenosine