Initial kinetics of the inactivation of calmodulin by the fungal toxin ophiobolin A.
Ophiobolin A, a fungal toxin that affects rice and maize, inhibits calmodulin by reacting with the lysine residues in calmodulin. Previous studies have shown that lysines 75, 77 and 148 in the calmodulin molecule were the binding sites for ophiobolin A, and that lysine 75 was the primary inhibitory site. In this study, we used kinetic analysis and mutated calmodulins to further characterize the inhibition process. The inhibition of bovine-brain calmodulin by ophiobolin A in the presence of excess ophiobolin A occurred rapidly and followed pseudo-first-order kinetics with a second-order rate constant of 3470 M(-1) min(-1). The kinetics data indicated that the binding of a single ophiobolin A molecule was enough to inactivate a calmodulin molecule. Mutant calmodulins in which two of the three aforementioned binding sites for ophiobolin A had been removed by site-directed mutagenesis were examined for the role of each of the three lysines in the inhibition. It was found that when lysine 75 or 77 in the mutant calmodulin was reacted with ophiobolin A, the resulting calmodulin became a poor activator of phosphodiestease. These results provide further evidence that lysine 75 in calmodulin is the primary inhibitory site for ophiobolin A.
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Department of Zoology, The University of Hong Kong, Pokfulam Road, Hong Kong, PR China.
S H Chick
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Leung
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PC
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001923:Brain Chemistry; D002147:Calmodulin / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D004789:Enzyme Activation; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D054830:Sesterterpenes; D013729:Terpenes / Q000378:metabolism / Q000494:pharmacology*
D002147:Calmodulin; D054830:Sesterterpenes; D013729:Terpenes; C040764:ophiobolin A; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D008239:Lysine