Atomistic and Systems-level Modeling of Phosphate Catalysis

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Phosphate processing enzymes are crucial to the majority of biochemical processes that all living cells depend on, including signal transduction, gene regulation, metabolism and energy transfer. The active sites of these enzymes are remarkably similar, in most cases requiring the presence of a catalytic Mg2+ ion bound to the phosphate groups, as the biological cofactor. However, there are distinct, specific differences in the coordination geometry of these enzymes that contain hidden information about the corresponding function. To gain insight towards constructing a general, unifying theory of how phosphate processing enzymes modulate the rates of their catalytic reactions, we propose to study two important systems. We will study the RAF kinase catalytic reaction. RAF kinases are part of the ERK signalling pathway, which is aberrantly activated in >30% of all cancers, with RAS and RAF being the main oncogenic factors. We will also study the catalytic reaction of a bacterial ligase, Ddl, from M. tuberculosis, the causative agent of TBC. Ddl is an essential enzyme in the biosynthesis of bacterial cell wall. It is one of the validated drug targets of the antibiotic drug Seromycine, and a major drug target against TBC. Seromycine is used as a second line of treatment, for multiple drug-resistant and extensively drug-resistant strains of M. tuberculosis.Using our novel computational methods that can provide accurate free energies and kinetic information about the dynamics of the systems, we will identify coupled proton transfer steps that occur together with the phosphate cleavage reactions. We will use our calculated structures and mechanism to develop and test novel inhibitor design strategies.