Specificity of Creatine Kinase for Guanidino Substrates

The specificity of rabbit muscle creatine kinase for a series of guanidino substrate analogs has been investigated at 0°. The maximum velocities range from ≤0.1 to 90% that of creatine whereas the Michaelis constants are as much as two orders of magnitude larger than that for creatine. The activity is found to be very sensitive to certain structural features of the guanidino substrate molecules, such as the length of the alkyl side chain length and the position of the carboxyl group. The dissociation constants, Kdiss, of the guanidino analogs from the abortive quaternary complex, MnADP-guanidino analog-enzyme, have been determined by using the difference in the proton relaxation rate of water in the presence of each respective quaternary complex and the ternary MnADP-enzyme complex. Kdiss was also determined for some of the analogs as an inhibition constant, Ki, for the reverse reaction. Nitrate is found to affect drastically both the dissociation constant of the analogs from their respective quaternary complexes and the proton relaxation rate of these complexes. For example, the Kdiss of creatine is decreased from 5 mm to 80 µm and the proton relaxation rate of water is decreased by 50% on the addition of 2 mm potassium nitrate. In the absence of nitrate the dissociation constants are generally of the same order as the Michaelis constants, while in the presence of nitrate they are between one and two orders of magnitude smaller. These results provide cogent evidence for the hypothesis that the nitrate anion acts as an analog of the planer phosphoryl group which is transferred, thus leading to a structure which resembles the transition state.