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Decavanadate inhibits catalysis by ribonuclease A
1Department of Biochemistry, University of Wisconsin-Madison 53706, USA.
Archives of Biochemistry and Biophysics
|October 6, 2000
Summary
Decavanadate inhibits bovine pancreatic ribonuclease A (RNase A) catalysis. This oxometalate binds RNase A with high affinity, similar to organo-vanadates, revealing a new class of enzyme inhibitors.
Area of Science:
- Biochemistry
- Enzymology
- Inorganic Chemistry
Background:
- Pentavalent organo-vanadates are established transition-state analogs for phosphoryl transfer.
- Bovine pancreatic ribonuclease A (RNase A) is a key enzyme in RNA processing.
Purpose of the Study:
- To investigate decavanadate as a potential inhibitor of RNase A.
- To characterize the binding affinity and interaction mechanism between decavanadate and RNase A.
Main Methods:
- Isothermal titration calorimetry (ITC) to determine binding affinity (Kd).
- Enzyme kinetics to measure inhibition of catalytic activity.
- Studies with site-directed mutagenesis to probe the role of active site residues.
Main Results:
- Decavanadate inhibits RNase A catalysis with a dissociation constant (Kd) of 1.4 microM.
- Binding involves a significant coulombic component, evidenced by reduced affinity with increasing NaCl concentration and weaker binding to mutant RNase A enzymes with altered cationic residues.
- Decavanadate exhibits binding affinity comparable to known pentavalent organo-vanadate inhibitors.
Conclusions:
- Decavanadate is the first identified oxometalate inhibitor of ribonuclease catalysis.
- The findings highlight decavanadate as a potent inhibitor of RNase A, with implications for understanding enzyme inhibition mechanisms and developing novel therapeutic agents.