A study of the kinetics of the reactions catalysed by arginine phosphokinase

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Abstract
The kinetics of the reaction, phosphoarginine + adenosine diphosphatearginine + adenosine triphosphate, catalyzed by arginine phosphokinase were studied. When enzymic activity is plotted against the total Mg2+ ion concentration, maximum activity is reached at a concentration of Mg2+ ions which is dependent on the concentration of ADP or ATP. When enzymic activity is plotted against the concentration of "free Mg2+ ion" (which is independent of the concentration of ADP or ATP) a Michaelis-Menten relation between enzymic activity and the concentration of Mg2+ and Mn2+ ions was demonstrated. The dissociation constants of the magnesium-arginine phosphokinase complex, determined under various conditions, were calculated from plots of 1/v against I/total Mg2+ ions and l/v against l/"free Mg2+ ions". The Km, values of the 4 substrates were determined. Those for ADP and ATP were calculated from plots of l/v against I/total substrate and l/v against l/"free substrate". The forward reaction was inhibited by ATP and adenylic acid; the reverse reaction was inhibited competitively by ADP. It was concluded that ADP and ATP are bound to the enzyme at the same site. There was competitive inhibition of the forward reaction by Ca2+ ions. The effect of temperature on the velocity of the forward and reverse reactions was studied and the apparent activation energies were calculated. The equilibrium of the reaction was influenced by pH and the concentration of Mg2+ ions. The conclusion was reached that Mg2+ ions react with arginine phosphokinase to form an active magnesium-arginine phosphokinase complex which in turn reacts with the free forms of ATP and ADP.