2019/11/15 by Claire Stines‐Chaumeil, François Mavré, Stines-Chaumeil, Claire +7
Biochemistry, Genetics and Molecular Biology · Materials Science · #Biomolecules (q-bio.BM) #Enzyme Catalysis and Immobilization #Enzyme Structure and Function #Enzyme function and inhibition #FOS: Biological sciences #Microbial metabolism and enzyme function
paper · pdf · doi:10.48550/arxiv.1911.06795
openalex publication_date 2019/11/15 · openalex created_date 2022/07/26 · openalex updated_date 2026/07/28
The ability to switch on the activity of an enzyme through its spontaneous\nreconstitution has proven to be a valuable tool in fundamental studies of\nenzyme structure/reactivity relationships or in the design of artificial signal\ntransduction systems in bioelectronics, synthetic biology, or bioanalytical\napplications. In particular, those based on the spontaneous\nreconstitution/activation of the apo-PQQ-dependent soluble glucose\ndehydrogenase (sGDH) from Acinetobacter calcoaceticus were widely developed.\nHowever, the reconstitution mechanism of sGDH with its two cofactors, i.e. the\npyrroloquinoline quinone (PQQ) and Ca2+, remains unknown. The objective here is\nto elucidate this mechanism by stopped-flow kinetics under single-turnover\nconditions. The reconstitution of sGDH exhibited biphasic kinetics,\ncharacteristic of a square reaction scheme associated to two activation\npathways. From a complete kinetic analysis, we were able to fully predict the\nreconstitution dynamic, but also to demonstrate that when PQQ first binds to\nthe apo-sGDH, it strongly impedes the access of Ca2+ to its enclosed position\nat the bottom of the enzyme binding site, thereby greatly slowing down the\nreconstitution rate of sGDH. This slow calcium insertion may purposely be\naccelerated by providing more flexibility to the Ca2+ binding loop through the\nspecific mutation of the calcium coordinating P248 proline residue, reducing\nthus the kinetic barrier to calcium ion insertion. The dynamic nature of the\nreconstitution process is also supported by the observation of a clear loop\nshift and a reorganization of the hydrogen bonding network and van der Waals\ninteractions observed in both active sites of the apo and holo forms, a\nstructural change modulation that was revealed from the refined X-ray structure\nof apo-sGDH (PDB:5MIN).\n