Kinetic Analyses of the Substrate Inhibition of Paramecium Arginine Kinase.

Paramecium tetraurelia expresses four types of arginine kinase (AK1-AK4). In a previous study, we showed that AK3 is characterized by typical arginine substrate inhibition, where enzymatic activity markedly decreases near a concentration of 1 mM of arginine substrate. This is in sharp contrast to the three other AK types, which obey the Michaelis-Menten reaction curve. Since cellular arginine concentration in another ciliate Tetrahymena is estimated to be 3-15 mM in vivo, Paramecium AK3 likely functions in conditions that are strongly affected by substrate inhibition. The purpose of this work is to find some novel aspect on the kinetic mechanism of the substrate inhibition of Paramecium AK3 enzyme. Substrate inhibition kinetics for AK3 were analyzed using three models and their validity were evaluated with three static parameters (R2, AICc, and Sy.x). The most accurate model indicated that not only ES but also the SES complex reacts to form products, the latter being the complex with two substrates in the active center. The maximum reaction rate for the SES complex, VmaxSES = 30.4 µmol Pi/min/mg protein, was one-eighth of the ES complex, VmaxES = 241.7. The dissociation constant for the SES complex (KiSES: 0.34 mM) was two times smaller than that of the ES complex (KsES: 0.61 mM), suggesting that after the primary binding of the arginine substrate (ES complex formation), the binding of a second arginine to the secondarily induced inhibitory site is accelerated to form an SES complex with a lower VmaxSES. The same kinetics were used for the S79A, S80A, and V81A mutants. The results indicate that the S79 residue is significantly involved in the process of binding the second arginine substrate. Herein, the KiSES value was ten times (3.62 mM) the value for the wild-type (0.34 mM), weakening substrate inhibition. In contrast, VmaxES and VmaxSES values for the mutants decreased by one-third, except for the VmaxSES of the S79A mutant, which had a value that was comparable with the value for the wild-type.