Mechanism of activation and inactivation of opsin: role of Glu113 and Lys296.
In previous studies, mutation of either Lys296 or Glu113 in bovine rhodopsin has been shown to result in constitutive activation of the apoprotein form, opsin [Robinson et al. (1992) Neuron 9, 719-725]. In this report, pH-rate profiles for the rhodopsin-catalyzed exchange of GTPgS for GDP on transducin are established for the constitutively active opsin mutants. All of the mutants, including the double-mutant E113Q,K296G, show a bell-shaped pH-rate profile. Therefore, it is evident that at least two ionizable groups in addition to Lys296 and Glu113 control the formation of the active opsin state. The sole effect of mutation at position 113 or 296 is to alter the ionization constant of the group with the higher pKa, called pka2. pKa2 decreases in the following order: rhodopsin/light (9.0) > K296E = K296G = E113Q,K296G (8.0) > E113Q (6.8) > K296H (6.6) >> wild-type opsin (< 5.0). These results are consistent with a model where activation of opsin involves (i) breaking of the salt bridge between Lys296 and Glu113, (ii) deprotonation of Lys296, and (iii) the net uptake of a proton from the solvent. Furthermore, exogenous addition of the chromophore all-trans-retinal shifts the wild-type and E113Q opsin equilibrium to favor the active state. In all these respects, the light-independent activation of the opsin mutants appears to proceed by a mechanism similar to that of light-activated rhodopsin.
Cohen
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GB
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.
Oprian
D D
DD
Robinson
P R
PR
D000818:Animals; D002417:Cattle; D002460:Cell Line; D003624:Darkness; D005973:Glutamine / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D012172:Retinaldehyde / Q000378:metabolism; D012243:Rhodopsin / Q000235:genetics / Q000378:metabolism; D017299:Rod Opsins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015293:Transducin / Q000378:metabolism
D017299:Rod Opsins; D005973:Glutamine; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006639:Histidine; D012243:Rhodopsin; D015293:Transducin; D008239:Lysine; D012172:Retinaldehyde