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The cooperativity effect in the reaction of soluble quinoprotein ( PQQ ‐containing) glucose dehydrogenase is not due to subunit interaction but to substrate‐assisted catalysis
Author(s) -
Duine Johannis A.,
Strampraad Marc J. F.,
Hagen Wilfred R.,
Vries Simon
Publication year - 2016
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.13829
Subject(s) - chemistry , cooperativity , substrate (aquarium) , dehydrogenase , protein subunit , cooperative binding , catalysis , enzyme , stereochemistry , biochemistry , biophysics , oceanography , gene , geology , biology
Soluble quinoprotein ( PQQ ‐containing) glucose dehydrogenase ( sGDH , EC 1.1.99.35 ) catalyzes the oxidation of β‐ d‐ glucose to d‐ glucono‐δ‐lactone. Although sGDH has many analytical applications, the relationship between activity and substrate concentration is not well established. Previous steady‐state kinetic studies revealed a negative cooperativity effect which has recently been ascribed to subunit interaction. To investigate this conclusion, stopped‐flow kinetic experiments were carried out on the reaction in which oxidized enzyme (E ox ) was reduced with substrates to E red . The appearance of E red is observed to be preceded by formation of an intermediate enzyme form, Int, which is mono‐exponentially formed from E ox . However, the rate of conversion of Int into E red depends hyperbolically on the concentration of substrate (leading to a 35‐fold stimulation in the case of glucose). Evidence is provided that substrate not only binds to E ox but also to Int and E red as well, and that the binding to Int causes the significant stimulation of Int decay. It is proposed that a proton shuffling step is involved in the decay, which is facilitated by binding of substrate to Int. Substituting the PQQ ‐activating Ca by a Ba ion lowered all reaction rates but did not change the stimulation factor. In summary, the previous proposal that the cooperativity effect of sGDH is due to interaction between its substrate‐loaded subunits is incorrect; it is due to substrate‐assisted catalysis of the enzyme. Enzymes EC 1.1.99.35 – soluble quinoprotein glucose dehydrogenase.