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The ability to switch on the activity of an enzyme through its spontaneousreconstitution has proven to be a valuable tool in fundamental studies ofenzyme structure/reactivity relationships or in the design of artificial signaltransduction systems in bioelectronics, synthetic biology, or bioanalyticalapplications. In particular, those based on the spontaneousreconstitution/activation of the apo-PQQ-dependent soluble glucosedehydrogenase (sGDH) from Acinetobacter calcoaceticus were widely developed.However, the reconstitution mechanism of sGDH with its two cofactors, i.e. thepyrroloquinoline quinone (PQQ) and Ca2+, remains unknown. The objective here isto elucidate this mechanism by stopped-flow kinetics under single-turnoverconditions. The reconstitution of sGDH exhibited biphasic kinetics,characteristic of a square reaction scheme associated to two activationpathways. From a complete kinetic analysis, we were able to fully predict thereconstitution dynamic, but also to demonstrate that when PQQ first binds tothe apo-sGDH, it strongly impedes the access of Ca2+ to its enclosed positionat the bottom of the enzyme binding site, thereby greatly slowing down thereconstitution rate of sGDH. This slow calcium insertion may purposely beaccelerated by providing more flexibility to the Ca2+ binding loop through thespecific mutation of the calcium coordinating P248 proline residue, reducingthus the kinetic barrier to calcium ion insertion. The dynamic nature of thereconstitution process is also supported by the observation of a clear loopshift and a reorganization of the hydrogen bonding network and van der Waalsinteractions observed in both active sites of the apo and holo forms, astructural change modulation that was revealed from the refined X-ray structureof apo-sGDH (PDB:5MIN).

Mechanism of reconstitution/activation of the soluble PQQ-dependent glucose dehydrogenase from Acinetobacter calcoaceticus: a comprehensive study