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 PRE-STEADY-STATE KINETICS OF BEEF HEART MITOCHONDRIAL ATPASE
Abstract
The pre-steady-state kinetics of beef heart mitochondrial ATPase (F(,1)) were examined. F(,1) was found to exhibit hysteretic behavior when hydrolyzing ATP. The hysteretic property was expressed as an activation process which occurred when the enzyme was mixed with its substrate, MgATP. The activation was usually completed before one full catalytic cycle was finished. This fact precluded the possibility that the activation occurred by an interaction at the catalytic site of the enzyme, such as an alternating site mechanism. Since the lag in hydrolysis increased as the concentration of enzyme increased, it was hypothesized that the activation was caused, at least in part, by the dissociation of some inhibitor from the enzyme. Since no protein dissociation phenomena have ever been observed with native F(,1), it was concluded that the dissociable inhibitor was a tightly bound nucleotide. Since the activation did not follow first order kinetics, the dissociation could not have been the only process involved in the activation. Other possibilities included multiple, kinetically distinct, nucleotide dissociations, enzyme conformational changes, or regulatory site interactions. The enzyme was apparently able to recognize nucleotide in a noncatalytic manner, as evidenced by the fact that F(,1) preincubated with ADP in the absence of substrate achieved partial activation (smaller lag times) before being introduced to substrate. ADP is also a time dependent inhibitor, exhibiting a slow hysteretic inhibition in addition to immediate competitive-like inhibition. The concentration of MgATP needed to cause the activation was concluded to lie somewhere below 0.05 mM, the lowest concentration used in these studies, since no effect on the activation was observed at this or higher ATP concentrations. This concentration was also less than the Km value of about 0.20 mM for catalysis observed in steady-state experiments. Magnesium caused no detectable kinetic effects at the concentrations used. Magnesium may only be required to form the MgATP complex, the actual substrate of F(,1).
Subject Area
Biochemistry
Recommended Citation
CLARK, DAVID DEAN, "THE PRE-STEADY-STATE KINETICS OF BEEF HEART MITOCHONDRIAL ATPASE" (1981). ETD collection for University of Nebraska-Lincoln. AAI8120158.
https://digitalcommons.unl.edu/dissertations/AAI8120158