A mechanistic role for protein oligomerization in the phosphagen kinase family

Abstract

Creatine and arginine kinases catalyze reversible phosphoryl transfer between ATP and creatine or arginine, respectively. Although these two kinases share approximately 80% sequence identity, the quaternary structure of arginine kinase (AK) is typically monomeric, whereas that of creatine kinase (CK) is dimeric or octomeric. Previous structural and functional studies of dimeric CK show that the binding of a transition state analogue complex [Cr-NO3-MgADP] by the two active sites displays negative cooperativity. The goal of this project is to determine whether protein oligomerization, specifically negative cooperativity, plays a functional role in the mechanism of these enzymes. Viscosity (trehalose) variation studies and pre-steady state kinetic analyses by rapid quench of the reactions catalyzed by wild-type dimeric CK, the R147A/R151A monomeric CK and an unusual dimeric AK from Stichopus japonicus, were conducted to examine the rate-determining step of each reaction. Collectively, the results show that the rate of the reaction catalyzed by the monomeric enzymes is limited by the rate of product release, whereas that of the dimeric enzymes is limited by phosphoryl transfer. Thus, negative cooperativity within the phosphagen kinase family enhances catalytic efficiency by decreasing the free energy barrier for product release. This work was supported by NSF grant #0344432.