Initial kinetics of the inactivation of calmodulin by the fungal toxin ophiobolin A

Ophiobolin A, a fungal toxin that affects rice and maize; inhibits calmodul in by reacting with the lysine residues in calmodulin. Previous studies hav e shown that lysines 75, 77 and 148 in the calmodulin molecule were the bin ding sites for ophiobolin A, and that lysine 75 was the primary inhibitory site. In this study, we used kinetic analysis and mutated calmodulins to fu rther characterize the inhibition process. The inhibition of bovine-brain c almodulin by ophiobolin A in the presence of excess ophiobolin A occurred r apidly and followed pseudo-first-order kinetics with a second-order rate co nstant 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 molec ule. Mutant calmodulins in which two of the three aforementioned binding si tes for ophiobolin A had been removed by site-directed mutagenesis were exa mined for the role of each of the three lysines in the inhibition. It was f ound that when lysine 75 or 77 in the mutant calmodulin was reacted with op hiobolin A, the resulting calmodulin became a poor activator of phosphodies tease. These results provide further evidence that lysine 75 in calmodulin is the primary inhibitory site for ophiobolin A. (C) 2000 Elsevier Science Ltd. All rights reserved.