Site-directed mutagenesis of the cGMP phosphodiesterase inhibitory gamma subunit from bovine rod outer segments: A new hypothesis on the mechanisms of catalytic subunits inhibition by gamma subunit and holoenzyme activation by transducin
Lipkin, V.M.; Alekseev, A.M.; Bondarenko, V.A.; Muradov, K., G.; Obukhov, A.N.; Zagranichnyi, V.E.
Bioorganicheskaya Khimiya 20(8-9): 821-832
Two mutants of the phosphodiesterase (PDE) gamma subunit (PDE-gamma) from bovine retinal rods were synthesized by sequential transcription and translation in vitro. PDE-gamma mutants R24E and H79L exhibited inhibitory properties similar to those of the wild-type PDE-gamma (wtPDE-gamma). At the same time, affinity to the rod outer segment (ROS) membranes is lower for R24E and higher for H79L in comparison with wtPDE-gamma. The transducin alpha subunit (in a complex with the GTP non-hydrolyzable analogue, GTP-gamma-S) activates the trypsin-treated PDE (tPDE) inhibited by wtPDE-gamma weaker than tPDE inhibited by R24E and stronger than tPDE inhibited by H79L. To explain the properties of these and earlier studied PDE-gamma mutants, a new hypothesis on the mechanisms of inhibition of the PDE catalytic subunit dimer (PDE-alpha-beta) by PDE-gamma and mechanism of the PDE holoenzyme (PDE-alpha-beta-gamma-2) activation by the transducin alpha subunit in a complex with GTP (T-alpha cntdot GTP) is proposed: 1) two sites on PDE-alpha-beta for the PDE-gamma binding (A- and the B-site) are different in structure. Sites on PDE-gamma interacting with A- and the B-sites on PDE-alpha-beta are also different in structure. The site on PDE-gamma interacting with the B-site partially overlaps with the T-alpha cntdot GTP binding site; 2) PDE-gamma bound to the B-site provides the main contribution to inhibition of the enzyme catalytic activity; 3) T-alpha cntdot GTP first interacts with the PDE-gamma bound to the A-site in the PDE holoenzyme and removes this PDE-gamma in a PDE-gamma cntdot (T-alpha cntdot GTP) complex. This results in a slight increase of the catalytic activity of the PDE-alpha-beta-gamma complex remaining bound to the ROS membranes; 4) after removal of PDE-gamma from the A-site, another T-alpha cntdot GTP molecule is enabled to interact with both PDE-alpha-beta and PDE-gamma bound to the B-site on PDE-alpha-beta. This interaction results in the formation of a ROS membrane-bound fully catalytically active triple complex PDE-alpha-beta cntdot PDE-gamma cntdot (T-alpha cntdot GTP).