Abstract
As part of the essential cellular system that maintains energy homeostasis, adenylate kinases (ADK) catalyze the interconversion of AMP and ATP to 2 ADPs. Fundamental work on the chemical process is done here to understand the mechanism and the energy associated with how the kinase catalyzes this specific reaction. The Arg150 residue of A. aeolicus ADK is thought to be directly involved in the process of phosphoryl transfer and stabilization of the transition state. The point mutation R150K was made and its crystal structure was solved. The structure showed that the lysine does not have contacts with the bound nucleotides and thus most likely cannot take part in the catalysis. The rate of the phosphoryl transfer in the wild type enzyme was at least 1000 times faster than in the R150K mutant. This shows that the interactions between the nucleotides and this residue in the protein are important in the minimization of the activation energy for chemical step. \r The second shell energy contribution to the phosphoryl transfer was explored through our studies of two ADK isoforms (thermophilic and mesophilic). They have significantly different phosphoryl transfer activities despite having identical active sites where the thermophile is 10 fold slower than the mesophile at 25°C. Through sequence and structural analysis of the two isoforms, we identified several residues in the second shell that may have significant contribution to ADK activity. We replaced one of the residues in question from the thermoADK with the corresponding residue from mesoADK. The resulting effect was explored through kinetic studies of the engineered mutant. We found that the mutant exhibited a 20% higher activity relative to the thermoADK. Although the effect of the mutation on the catalytic efficiency of ADK was in the direction we predicted, its magnitude was less than expected.\r Additionally, the thermo-stability evolutionary factor was investigated with ADK by measuring the temperature dependence of their overall catalysis. Our preliminary data indicate that ADKs decrease their enthalpic contributions in the catalysis, consistent with the theory that enzymes become less dependent on temperature as they evolve with the earth cooling in time.