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Binding of NAD+ by cholera toxin
T S Galloway1, S van Heyningen
1Department of Biochemistry, University of Edinburgh, U.K.
The Biochemical Journal
|May 15, 1987
Summary
Cholera toxin acts as an NAD+ glycohydrolase with a high Km for NAD+. Its binding affinity (Kd) was accurately determined to be approximately 4 mM using tryptic digestion inhibition assays.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Cholera toxin possesses NAD+ glycohydrolase activity.
- Understanding the kinetics and binding properties of this enzyme is crucial for elucidating its mechanism of action.
Purpose of the Study:
- To determine the Michaelis constant (Km) for NAD+ of cholera toxin as an NAD+ glycohydrolase.
- To characterize the dissociation constant (Kd) of NAD+ binding to cholera toxin.
- To identify competitive inhibitors of the NAD+ glycohydrolase reaction.
Main Methods:
- Enzyme kinetics assays measuring NAD+ glycohydrolase activity.
- Equilibrium dialysis to estimate dissociation constants.
- Proteolytic digestion assays (tryptic digestion of A1 peptide) combined with densitometry to determine Kd.
Main Results:
- The Km for NAD+ was found to be 4 mM, increasing to approximately 50 mM in the presence of ADP-ribose acceptors.
- Equilibrium dialysis suggested a Kd of approximately 3 mM with one binding site.
- Tryptic digestion inhibition assays provided a more accurate Kd of 4.0 ± 0.4 mM.
- Competitive inhibitors were identified as molecules retaining the adenine and nicotinamide moieties of NAD+.
Conclusions:
- Cholera toxin exhibits a high Km for NAD+, indicating a specific binding requirement.
- The dissociation constant (Kd) for NAD+ is approximately 4 mM.
- Structural analogues of NAD+ that mimic its key components can act as competitive inhibitors.