[Oligonucleotide-directed mutagenesis of inhibitory gamma-subunits of cGMP phosphodiesterase from bovine outer rod segments. A new hypothesis on mechanisms for inhibiting catalytic subunits by gamma-subunits and activation of a holoenzyme by transducin]

Abstract

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.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.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.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.(T alpha.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.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.PDE gamma.(T alpha.GTP).