Abstract
By using 31P double-resonance nucelar magnetic resonance, preliminary kinetic measurements were made on the equilibrium reaction 2ADP in equilibrium ATP +AMP catalyzed by adenylate kinase. The double-resonance method used consisted of selectively inverting the spin population in a particular chemical environment and observing the transfer of the inverted spin to another chemical environment. The chemical transfer time between AMP and ADP, free in solution, was proportional to the substrate-to-enzyme ratio, and a transfer rate of 95 mol of AMP/see per mol of enzyme was obtained. In the same series of experiments, the life-time of AMP in solution was determined from the extra broadening of its resonance line due to the active enzyme. This gave a rate 3.2 times faster than the overall transfer rate given above. From these rates and other nuclear magnetic resonance measurements wehave calculated the individual rate constants between the ternary complexes, AMP-enzyme-ATP and ADP-enzyme-ADP. In addition, we obtained one of the ADP off-rates from this latter complex. The rates on the enzyme are approximately 1250 and 500mol/sec per mol of enzyme for the forward and reverse directions, respectively. The ADP has an off-rate of 450 sec (-1).
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