The cooperativity effect in the reaction of soluble quinoprotein (PQQ‐containing) glucose dehydrogenase is not due to subunit interaction but to substrate‐assisted catalysis

• Published in: The FEBS journal 2016-10, Vol.283 (19), p.3604-3612
• Main Authors: Duine, Johannis A., Strampraad, Marc J. F., Hagen, Wilfred R., Vries, Simon
• Format: Article
• Language: English
• Subjects: Barium - chemistry; Biocatalysis; Catalysis; Dehydrogenases; Enzymes; Glucose - metabolism; glucose dehydrogenase; Glucose Dehydrogenases - chemistry; Glucose Dehydrogenases - metabolism; Kinetics; PQQ Cofactor - chemistry; presteady‐state kinetics; Protein Subunits - chemistry; Protein Subunits - metabolism; pyrroloquinoline quinone; singular value decomposition; substrate‐assisted catalysis
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.13829

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 (Eox) was reduced with substrates to Ered. The appearance of Ered is observed to be preceded by formation of an intermediate enzyme form, Int, which is mono‐exponentially formed from Eox. However, the rate of conversion of Int into Ered 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 Eox but also to Int and Ered 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. Quinoprotein glucose dehydrogenase shows unusual kinetics in the oxidation of d‐β‐glucose to d‐glucono‐δ‐lactone, previously reported to result from subunit interaction. In stopped‐flow experiments, analyzed with the method of singular value decomposition, it is now found that substrate binding to an enzyme intermediate (Int) accelerates the decomposition of Int. This substrate‐assisted catalysis fully explains the kinetics without subunit interaction.