Is the ATP analogue adenosine 5′‐sulfatopyrophosphate an alternative substrate or inhibitor of creatine kinase?

Enzymes are proficient catalysts and show high levels of substrate selectivity and stereospecificity; recent observations suggest that enzymes may also posses the ability to catalyze a reaction distinct from its standard biological reaction. That behavior is termed catalytic promiscuity. Here we investigated the possibility that creatine kinase (CK), a physiologically important enzyme that normally catalyzes reversible phosphoryl transfer between creatine and MgATP, is also able to catalyze sulfuryl transfer between these two substrates using an ATP analogue, adenosine 5′‐sulfatopyrophosphate (ADPSO 3 ). ADPSO 3 was successfully synthesized and purified by ion exchange chromatography, as verified by 31 P NMR. The resulting products of potential sulfuryl transferase activity by CK were analyzed by HPLC‐UV at 254 nm. Incubation of creatine, Mg‐ or Mn‐ADPSO 3 with CK at pH 9.5 and 30 °C for 27–45 hours did not result in any detectable sulfuryl transferase activity. Over that same period of time, ADPSO 3 was found not to undergo any significant spontaneous or enzyme‐catalyzed hydrolysis. Hence, these data suggest that ADPSO 3 is an unreactive substrate analogue and therefore useful for determining the crystal structure of the CK‐creatine‐ADPSO 3 complex. This research was supported by NSF grant #0344432.