SITE-DIRECTED MUTAGENESIS OF THE CGMP PHO SPHODIESTERASE 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

Two mutants of the phosphodiesterase (PDE) gamma subunit (PDEgamma) fr om bovine retinal rods were synthesized by sequential transcription an d translation in vitro. PDEgamma mutants R24E and H79L exhibited inhib itory properties similar to those of the wild-type PDEgamma (wtPDEgamm a). At the same time, affinity to the rod outer segment (ROS) membrane s is lower for R24E and higher for H79L in comparison with wtPDEgamma. The transducin alpha subunit (in a complex with the GTP non-hydrolyza ble analogue, GTPgammaS) activates the trypsin-treated PDE (tPDE) inhi bited by wtPDEgamma weaker than tPDE inhibited by R24E and stronger th an tPDE inhibited by H79L. To explain the properties of these and earl ier studied PDEgamma mutants, a new hypothesis on the mechanisms of in hibition of the PDE catalytic subunit dimer (PDEalphabeta) by PDEgamma and mechanism of the PDE holoenzyme (PDEalphabetagamma2) activation b y the transducin alpha subunit in a complex with GTP (Talpha.GTP) is p roposed: 1) two sites on PDEalphabeta for the PDEgamma binding (A- and the B-site) are different in structure. Sites on PDEgamma interacting with A- and the B-sites on PDEalphabeta are also different in structu re. The site on PDEgamma interacting with the B-site partially overlap s with the Talpha.GTP binding site; 2) PDEgamma bound to the B-site pr ovides the main contribution to inhibition of the enzyme catalytic act ivity; 3) Talpha.GTP first interacts with the PDEgamma bound to the A- site in the PDE holoenzyme and removes this PDEgamma in a PDEgamma.(Ta lpha.GTP) complex. This results in a slight increase of the catalytic activity of the PDEalphabetagamma complex remaining bound to the ROS m embranes; 4) after removal of PDEgamma from the A-site, another Talpha .GTP molecule is enabled to interact with both PDEalphabeta and PDEgam ma bound to the B-site on PDEalphabeta. This interaction results in th e formation of a ROS membrane-bound fully catalytically active triple complex PDEalphabeta.PDEgamma.(Talpha.GTP).