Abstract
The kinetic mechanism and evaluation of several potential inhibitors of purified arginine kinase from the cockroach (Periplanta americana) were investigated. This monomeric phosphagen kinase is important in maintaining ATP levels during the rapid energy demands of muscle required for contraction and motility. Analysis reveals the following dissociation constants (mM) for the binary complex: E·Arg˜P → E + Arg˜P, K = 1.0; E·Arg → E + Arg, K = 0.45; E·MgATP → E + MgATP, K = 0.17; E·MgADP → E + MgADP, K = 0.12; and the ternary complex: Arg˜P·E·MgADP → E·MgADP + Arg˜P, K = 0.94; Arg·E·MgATP → E·MgATP + Arg, K = 0.49; MgATP·Enz·Arg → E·Arg +MgATP, K = 0.14; MgADP·E·Arg˜P → E·Arg˜P + MgADP, K = 0.09. For a particular substrate, the ratio of the dissociation constants for the binary to ternary complex is close to one, indicating little, if any, cooperativity in substrate binding for the rapid equilibrium, random addition mechanism. The time course of the arginine kinase reaction exhibits a pronounced curvature, which, as described for enzyme from other sources, is attributed to formation of an inhibitory catalytic dead‐end complex, MgADP·E·Arg. The curvature is accentuated by the addition of monovalent anions, including borate, thiocyanate, and, most notably, nitrite and nitrate. This effect is attributed to stabilization of the dead‐end complex through formation of a transition state analog. However, the substantial decrease in initial velocity (92%) caused by nitrate is due to an additional inhibitory effect, further characterized as non‐competitive inhibition (K~i~ = 8.0 mM) with the substrate L‐arginine. On the other hand, borate inhibition of the initial velocity is only 30% with significant subsequent curvature, suggesting that this anion functions as an inhibitor mainly by formation of a transition state analog. However, some component of the borate inhibition appears to be mediated by an apparent partial competitive inhibition with L‐arginine. D‐arginine is not a substrate for arginine kinase from the cockroach, but is an effective competitive inhibitor with a K~i~ = 0.31 mM. L‐Canavanine is a weak substrate for arginine kinase (K~m~ = 6.7 mM) with a V~max~ for the pure enzyme that is approximately one‐third that of L‐arginine. However, initial velocity experiments of substrate mixtures suggest that competition between L‐canavanine and L‐arginine may not be a simple summation effect and may involve a structural modification. Sensitivity of arginine kinase activity to D‐arginine as well as nitrate and borate anions, coupled with the fact that L‐arginine is an essential amino acid for the cockroach, suggest that arginine kinase could be a useful chemotherapeutic target for the control of cockroach proliferation. Arch. Insect Biochem. Physiol. 57:166–177, 2004. © 2004 Wiley‐Liss, Inc.