Interaction of Manganous Ion, Substrates, and Anions with Arginine Kinase: MAGNETIC RELAXATION RATE STUDIES OF WATER PROTONS AND KINETIC ANION EFFECTS

Abstract

Abstract 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(II) 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 l- and d-arginine in the quaternary complex also have been examined. Manganous ion binds to arginine kinase at approximately two sites with a Kd value of 0.55 mm, and the PRR enhancement of the binary complex, eb, at 24.3 MHz and 25° is 9.6. MnADP binds quite strongly to the enzyme, K2 = 7.5 µm, and the resulting ternary complex has a highly enhanced PRR, et = 18.9. Although MnATP binds more weakly to the enzyme than MnADP by an order of magnitude, K2 = 79 µm, 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 quaternary complex which has a PRR value that is approximately 50% of the value for the ternary complex. The dissociation constant, Kdiss, for l-arginine in the quaternary complex, 0.15 mm, 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 quaternary complex containing d-arginine is within 10% of the value of the l-arginine-containing complex. The addition of 10 mm nitrate produces a 20-fold decrease in the dissociation constant for d- or l-arginine from the quaternary complex. The dissociation constant of nitrate from the complex with l-arginine (Kdiss = 0.06 mm) is increased 50-fold upon substitution of d-arginine, and nitrate produces a decrease of 48% in the PRR of the quaternary complex in the presence of l-arginine and of 36% in the presence of d-arginine. The planar anions, thiocyanate, nitrite, and formate, lower the PRR of the quaternary complex to a small extent. However, the tetrahedral anions, sulfate, perchlorate, and trichloroacetate, are ineffective in this respect. Nitrate both lowers the PRR to a greater extent and binds about 2 orders of magnitude more tightly than any of the other anions. In addition, nitrate inhibits the forward reaction of arginine kinase, produces a marked curvature in the reaction progress curve, and augments the inhibition by MgADP. Apparently this anion locks the enzyme into a transition-state analog complex in which all components are bound very tightly. The effects of nitrate on arginine kinase complexes are quite similar to the anion's effects on creatine kinase complexes. The results for both enzymes are consistent with the hypothesis that the nitrate anion acts as an analog of the transferable phosphoryl group in its trigonal bipyramidal configuration in the transition state. These observations lend further support to the contention that basic aspects of the catalytic mechanism are similar for both guanidino kinases.