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THE CHEMISTRY OF CHROMIUM(III)-NUCLEOTIDES AND THEIR INTERACTION WITH BEEF HEART MITOCHONDRIAL F1-ATPASE
Abstract
The separation of the four diastereomers of (beta),(gamma)-bidentate CrATP, and the two diastereomers of (alpha),(beta)-bidentate CrADP by reverse-phase HPLC techniques is described. This technique provides complete resolution of the diastereomers from both compounds within 10 minutes and uses methanesulfonate and esthanesulfonate as the ion-pairing agents. Circular dichroism spectra of all diastereomers were obtained, substrate, and inhibition specificities were examined, and the screw sense of each isomer was then identified according to the assignments made by Dunaway-Mariano and Cleland Dunaway-Mariano, D., and Cleland, W. W. (1980) Biochemistry 19, 1506-1515. By use of the described separation techniques, a scheme was devised for monitoring the interconversion of the (alpha),(beta)-bidentate CrADP diastereomers under various conditions of pH, temperature, buffer type, and buffer concentration. Rate constants were calculated using interative descent methods, and a mechanism proposed from the results. The data suggest that specific base catalysis is involved and that an S(,N)1CB (substitution, nucleophilic, unimolecular, conjugate base) type mechanism is most probable. The synthesis and isolation of the (gamma)-monodentate and (beta),(gamma)-bidentate CrITP complexes are described. These two chelation isomers, as well as the isolated diastereomers of (alpha),(beta)-bidentate CrADP and (beta),(gamma)-bidentate CrATP were tested as inhibitors of the F(,1)-ATPase and F(,1)-ITPase activities. F(,1) shows no binding preference with the isolated diastereomers of (beta),(gamma)-bidentate CrATP, and is inhibited by the (DELTA),(alpha),(beta)-bidentate CrADP diastereomer only slightly better than the (LAMDA) epimer. Gamma-monodentate CrITP was a weak inhibitor of both the ATPase and ITPase activities, whereas (beta),(gamma)-bidentate CrITP failed to inhibit either activity up to a concentration of 3.2 mM. MgATP(beta)S (S(,p)) and CdATP(beta)S (R(,p)), which have the (DELTA)-chelate structure, were slightly better substrates than their R(,p), S(,p) counterparts. The large activation energy observed for the isomers of CdATP(beta)S was not observed for other metal-nucleotide of metal-phosphorothioate nucleotide complexes. The hydrolysis of CdATP(gamma)S showed a value of V(,m) which was the largest of all the metal-phosphorothioate nucleotide complexes, while that for MgATP(gamma)S was the smallest.
Subject Area
Biochemistry
Recommended Citation
GRUYS, KENNETH JAMES, "THE CHEMISTRY OF CHROMIUM(III)-NUCLEOTIDES AND THEIR INTERACTION WITH BEEF HEART MITOCHONDRIAL F1-ATPASE" (1984). ETD collection for University of Nebraska-Lincoln. AAI8423787.
https://digitalcommons.unl.edu/dissertations/AAI8423787