Phosphorothioate analogs of ATP as the substrates of dynein and ciliary or flagellar movement.

The phosphorothioate analog of ATP has a sulfur atom replacing a non-bridging oxygen atom of the triphosphate moiety of ATP. Due to the tetrahedral nature of the phosphorus atom, stereoisomers are known to exist, designated as the S_p and R_p isomers. We have reported [Shimizu & Furusawa (1986) Biochemistry 25, 5787] on the hydrolytic activity of the 22S dynein from Tetrahymema cilia towards the phosphorothioate analogs of ATP. In this paper, we extend our study and report on the microtubule-dynein dissociation by these analogs and on their ability to support sea urchin flagellar dynein enzymatic activity as well as ciliary or flagellar motility. It has been shown that the microtubules — 22s-dyein complex is dissociated by the binding of ATP to the enzymatic sites [Porter & Johnson (1983) J. Biol. Chem. 258, 6575]. We studied the dissociation by adenosine 5’-[α-thio]triphosphate (ATP[αS]), S_p and R_p, by light-scattering stopped-flow methods. The dissociation by (S_p)ATP[αS] was rapid and the rate of the light-scattering change by (S_p)ATP[αS] was a hyperbolic function of the nucleotide concentration, indicating that dissociation was a two-step process. On the other hand, (R_p )ATP[αS] up to 2 mM induced only slow and partial dissociation of the complex, while, in the presence of vanadate, it induced complete dissociation with a slightly higher rate (0.5 s^-1). The adenosine 5’-[β-thio]triphosphate (ATP[βS]) isomers did not induce dissociation. The hydrolytic activity of the outer arm dyein from sea urchin sperm flagella towards these analogs was similar to that of 22S dynein. The ratios of V_max (nmol · mg protein^-1 · min^-1)/apparent K_m (µM) of this dynein were 400-720, 53, 9.7, 0.62 and 0.028 for ATP, ATP[αS] (S_p or R_p), ATP[βS] (S_p or R_p), respectively, in the presence of Mg²⁺ as the supporting cation. This dynein exhibited the same stereospecificity at β phosphate as the 22S dynein or myosin. The detergent models of Tetrahymena or sea urchin spermatozoa were reactivated only by ATP or (S_p)ATP[αS] while other analogs were ineffective. The maximal beat frequency of the cilia or flagella reactivated by (S_p)ATP[αS] was one‐quarter to one‐half of that produced by ATP reactivation. [ABSTRACT FROM AUTHOR]