Binding interactions in a kinase active site modulate background ATP hydrolysis.

ABSTRACT

Kinases play central roles in many cellular processes, transferring the terminal phosphate groups of nucleoside triphosphates (NTPs) onto substrates. In the absence of substrates, kinases can also hydrolyse NTPs producing NDPs and inorganic phosphate. Hydrolysis is usually much less efficient than the native phosphoryl transfer reaction. This may be related to the fact that NTP hydrolysis is metabolically unfavorable as it unproductively consumes the cell's energy stores. It has been suggested that substrate interactions could drive changes in NTP binding pocket, activating catalysis only when substrates are present. Structural data show substrate-induced conformational rearrangements, however there is a lack of corresponding functional information. To better understand this phenomenon, we developed a suite of isothermal titration calorimetry (ITC) kinetics methods to characterize ATP hydrolysis by the antibiotic resistance enzyme aminoglycoside-3'-phosphotransferase-IIIa (APH(3')-IIIa). We measured Km, kcat, and product inhibition constants and single-turnover kinetics in the presence and absence of non-substrate aminoglycosides (nsAmgs) that are structurally similar to the native substrates. We found that the presence of an nsAmg increased the chemical step of cleaving the ATP γ-phosphate by at least 10- to 20-fold under single-turnover conditions, supporting the existence of interactions that link substrate binding to substantially enhanced catalytic rates. Our detailed kinetic data on the association and dissociation rates of nsAmgs and ADP shed light on the biophysical processes underlying the enzyme's Theorell-Chance reaction mechanism. Furthermore, they provide clues on how to design small-molecule effectors that could trigger efficient ATP hydrolysis and generate selective pressure against bacteria harboring the APH(3')-IIIa.