Phosphoenzyme conformational states and nucleotide-binding site hydrophobicity following thiol modification of the Ca2+-ATPase of sarcoplasmic reticulum from skeletal muscle.
Enhanced fluorescence of the ATP analogue 2',3'-O-(2,4,6-trinitrocyclohexyldienylidine)adenosine 5'-triphosphate (TNP-ATP), bound to the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum, is closely related to phosphoenzyme levels (Bishop, J. E., Johnson, J. D., and Berman, M. C. (1984) J. Biol. Chem. 259, 15163-15171) and has an emission maximum consistent with decreased polarity of the TNP-ATP-binding site. The phosphoenzyme conformation responsible for increased nucleotide-binding site hydrophobicity has been studied by redistribution of phosphoenzyme intermediates following specific thiol group modification. N-Ethylmaleimide, in the presence of 50 microM Ca2+, 1 mM adenyl-5'-yl imidodiphosphate, pH 7.0, at 25 degrees C for 30 min, selectively modified the SH group essential for phosphoenzyme decomposition, which resulted in decreased ATPase activity, Ca2+ uptake, and a decrease in ATP-induced TNP-ATP fluorescence. Phosphorylated (Ca2+, Mg2+)-ATPase levels from [gamma-32P] ATP remained relatively unaffected (3.1 nmol/mg), but the ADP-insensitive fraction decreased from 56 to 15%. Phosphoenzyme levels from 32Pi were also decreased to the same extent as turnover, with equivalent loss of Pi-induced TNP-ATP fluorescence. The E1 to E2 transition, as monitored by the change in intrinsic tryptophan fluorescence, was unaffected. Modification of thiol groups of unknown function did not modify turnover-induced TNP-ATP fluorescence. It is concluded that the ADP-insensitive phosphoenzyme, E2-P, is responsible for enhanced TNP-ATP fluorescence. This suggests that the conformational transition, 2Ca2+outE1 approximately P----2Ca2+inE2-P, is associated with altered properties of the noncatalytic, or regulatory, nucleotide-binding site.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D009711:Nucleotides / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D009711:Nucleotides; D010750:Phosphoproteins; D013438:Sulfhydryl Compounds; D000266:Adenylyl Imidodiphosphate; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D005033:Ethylmaleimide