Molecular mechanism of ATP versus GTP selectivity of adenylate kinase

Enzymatic substrate selectivity is critical for the precise control of metabolic pathways. In cases where chemically related substrates are present inside cells, robust mechanisms of substrate selectivity are required. Here, we report the mechanism utilized for catalytic ATP versus GTPselectivity during adenylate kinase( Adk) -mediated phosphorylation of AMP. Using NMR spectroscopy we found that while Adkadopts a catalytically competent and closed structural state in complex with ATP, the enzyme is arrested in a catalytically inhibited and open state in complex with GTP. X-ray crystallography experiments revealed that the interaction interfaces supporting ATP and GTPrecognition, in part, are mediated by coinciding residues. The mechanism provides an atomic view on how the cellular GTPpool is protected from Adkturnover, which is important because GTPhas many specialized cellular functions. In further support of this mechanism, a structure–function analysis enabled by synthesis of ATP analogs suggests that a hydrogen bond between the adenine moiety and the backbone of the enzyme is vital for ATP selectivity. The importance of the hydrogen bond for substrate selectivity is likely general given the conservation of its location and orientation across the family of eukaryotic protein kinases.