Off-campus UNL users: To download campus access dissertations, please use the following link to log into our proxy server with your NU ID and password. When you are done browsing please remember to return to this page and log out.
Non-UNL users: Please talk to your librarian about requesting this dissertation through interlibrary loan.
THE EFFECT OF SUBSTRATE ANALOGS ON THE KINETICS AND REGULATION OF BEEF HEART MITOCHONDRIAL F(1)-ATPASE
Abstract
Bidentate cobalt(III)tetraamine adenosine triphosphate was a mixed noncompetitive inhibitor of F$\sb 1$-catalyzed ITP hydrolysis and ATP hydrolysis in the absence and presence of bicarbonate. Co(NH$\sb 3)\sb 4$ATP was used in dual inhibitor studies with adenylylimidodiphosphate and azide; it was mutually exclusive with both. The compound also protected F$\sb 1$ from inactivation by 4-chloro-7-nitrobenzofurazan. Mono and bidentate Cr(NH$\sb 3)\sb 4$ATP, and ${\rm Rh(H\sb 2 O)\sb{n}ATP}$ were also mixed noncompetitive inhibitors of F$\sb 1$-catalyzed ATP hydrolysis. These compounds were used in dual inhibition experiments, along with mono and bidentate CrATP, and Co(NH$\sb 3)\sb 4$ATP. All exchange-inert metal nucleotides examined were mutually exclusive inhibitors of F$\sb 1$. It is postulated that the pK$\sb{\rm a}$ of the metal coordinated ligands is related to the potency of inhibition by these compounds. It appears probable exchange-inert nucleotide complexes are binding to site(s) in addition to the catalytic site(s) of F$\sb 1$. The $3\sp\prime$- O -(4-benzoyl)benzoyl derivatives of ATP (BzATP) and ITP (BzITP) were synthesized and tested as photoaffinity labels of F$\sb 1$. In the absence of actinic light, both BzATP and BzITP are substrates of F$\sb 1$, therefore BzATP and BzITP bind to the catalytic site(s) of F$\sb 1$. BzATP inactivates F$\sb 1$ with pseudo-first order kinetics, while inactivation of F$\sb 1$ by BzITP follows biphasic kinetics. BzATP is effective at lower concentrations than BzITP. Differential inactivation of F$\sb 1$-catalyzed ATP and ITP hydrolysis is observed for both benzophenone-derivatized nucleotides. Since enzyme photolyzed with BzITP is more inactivated for ATP hydrolysis than it is for ITP hydrolysis, and the reverse is true for enzyme photolyzed with BzATP, it is clear active enzyme exists with photoaffinity label covalently bound. This suggests that BzATP and BzITP both bind at two sites on F$\sb 1$ and the enzyme communicates between sites the identity of the nucleotide bound. The differential inactivation of F$\sb 1$ ATP and ITP hydrolysis is explained by a model which demands symmetrical occupation of regulatory sites for enzyme activity.
Subject Area
Biochemistry
Recommended Citation
STEINKE, LAUREY ANNE, "THE EFFECT OF SUBSTRATE ANALOGS ON THE KINETICS AND REGULATION OF BEEF HEART MITOCHONDRIAL F(1)-ATPASE" (1987). ETD collection for University of Nebraska-Lincoln. AAI8719787.
https://digitalcommons.unl.edu/dissertations/AAI8719787