Estimation of the rate constants associated with the inhibitory effect of okadaic acid on type 2A protein phosphatase by time-course analysis.
As is often the case with tightly binding inhibitors, okadaic acid produces its inhibitory effect on type 2A protein phosphatase (PP2A) in a time-dependent manner. We measured the rate constants associated with the binding of okadaic acid to PP2A by analysing the time-course of the reduction of the p-nitrophenyl phosphate (pNPP) phosphatase activity of the enzyme after application of okadaic acid. The rate constants for dissociation of okadaic acid from PP2A were also estimated from the time-course of the recovery of the activity from inhibition by okadaic acid after addition of a mouse IgG1 monoclonal antibody raised against the inhibitor. Our results show that the rate constants for the binding of okadaic acid and PP2A are of the order of 10(7) M-1.s-1, a typical value for reactions involving relatively large molecules, whereas those for their dissociation are in the range 10(-4)-10(-3) s-1. The very low values of the latter seems to be the determining factor for the exceedingly high affinity of okadaic acid for PP2A. The dissociation constants for the interaction of okadaic acid with the free enzyme and the enzyme-substrate complex, estimated as the ratio of the rate constants, are both in the range 30-40 pM, in agreement with the results of previous dose-inhibition analyses.
Takai
A
A
Department of Physiology, School of Medicine, Nagoya University, Japan.
Ohno
Y
Y
Yasumoto
T
T
Mieskes
G
G
D000818:Animals; D000911:Antibodies, Monoclonal; D004988:Ethers, Cyclic / Q000276:immunology / Q000494:pharmacology*; D066298:In Vitro Techniques; D007700:Kinetics; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D011817:Rabbits
D000911:Antibodies, Monoclonal; D004988:Ethers, Cyclic; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases