This study is concerned with the development of kinetic-based bioaffinity chromatographic systems for purification of ATP-dependent kinases, with a particular focus on the allosteric yeast hexokinase enzyme (EC 126.96.36.199). Synthesis and characterization of highly substituted N(6)-linked and S(6)-linked immobilized ATP derivatives are described using a rapid solid-phase modular approach. Evaluation of the new immobilized ATP derivatives has been carried out using model chromatographic studies with yeast hexokinase, employing specific substrate analogues (N-acetyl-D-glucosamine and suramin) to promote biospecific adsorption, in the presence and absence of citrate (a so-called allosteric activator of hexokinase activity). In this paper, successful bioaffinity chromatography systems were developed for yeast hexokinase and, as a result, interesting binding and catalytic properties of the enzyme were highlighted and explored. The overall results confirm the potential for extrapolation of the kinetic locking-on tactic, a general kinetic-based bioaffinity approach already developed for the NAD(P)(+)-dependent dehydrogenases, to ATP/ADP-dependent enzymes. However, in view of the enhancement of the intrinsic ATPase activity of hexokinase with glucosamine derivatives, and the coincidental hydrolysis of immobilized ATP to immobilized ADP, future developments necessary to support adaptation of the approach to ATP-dependent enzymes are discussed.
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