Interaction of manganous ion, substrates, and anions with arginine kinase. Magnetic relaxation rate studies of water protons and kinetic anion effects.

The interactions of ADP and the arginine enantiomers with arginine kinase have been investigated by measuring the enhancement of the longitudinal water proton relaxation rate (PRR) due to the paramagnetic Mn(I1) ion in the enzymesubstrate complexes. Anion effects on the kinetics of the forward reaction of arginine kinase, on the PRR of quaternary arginine kinase-MnADP-arginine complexes, and on the binding of Land D-arginine in the quaternary complex also have been examined. Manganous ion binds to a&nine kinase at approximately two sites with a K. value of 0.55 mu, and the PRR enhancement of the binary complex, fb, at 24.3 MHz and 25” is 9.6. MnADP binds quite strongly to the enzyme, KZ = 7.5 PM, and the resulting ternary complex has a highly enhanced PRR, e: = 18.9. Although MnATP binds more weakly to the enzyme than MnADP by an order of magnitude, K2 = 79 PM, the enhancement factor for the ATP ternary complex, et = 16.7, is within 12% of the value for the ADP ternary complex. Free ADP binds about 7 times more weakly to the enzyme than does MnADP, whereas the binding of ATP is only slightly weaker than that of MnATP. The substrate L-arginine binds to the ternary E-MnADP complex forming an abortive quatemary complex which has a PRR value that is approximately 50% of the value for the ternary complex. The dissociation con&auf, Kdiss, for r.-arginine in the quaternary complex, 0.15 mu, was determined from titration data using PRR as the parameter. Although D-arginine binds to the ternary complex about 300 times more weakly than L-arginine, the PRR value of the quatemary complex containing D-arginine is within 10% of the value of the L-arginine-containing complex. The addition of 10 mrd nitrate produces a ZO-fold decrease in the dissociation constant for Dor ~-a@nine from the quatemary complex. The dissociation constant of nitrate from the complex with L-arginine (KdiSS = 0.06 mu) is increased SO-fold upon substitution of D-arginine, and