title: The cooperativity effect in the reaction of soluble quinoprotein (PQQ-containing) glucose dehydrogenase is not due to subunit interaction but to substrate-assisted catalysis.
abstract: 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 Eredis 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 Ereddepends 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 Eoxbut also to Int and Eredas 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.
  
  
  
  EC 1.1.99.35 - soluble quinoprotein glucose dehydrogenase.
authors: Department of Biotechnology, Delft University of Technology, The Netherlands. j.a.duine@tudelft.nl.
authors: Johannis A
authors: JA
authors: Duine
authors: Department of Biotechnology, Delft University of Technology, The Netherlands.
authors: Marc J F
authors: MJ
authors: Strampraad
authors: Department of Biotechnology, Delft University of Technology, The Netherlands.
authors: Wilfred R
authors: WR
authors: Hagen
authors: Department of Biotechnology, Delft University of Technology, The Netherlands.
authors: Simon
authors: S
authors: de Vries
mesh_terms: D001464:Barium / Q000737:chemistry; D055162:Biocatalysis; D005947:Glucose / Q000378:metabolism; D005948:Glucose Dehydrogenases / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D045542:PQQ Cofactor / Q000737:chemistry; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism
chemical_list: D021122:Protein Subunits; D001464:Barium; D045542:PQQ Cofactor; D005948:Glucose Dehydrogenases; D005947:Glucose
keywords: glucose dehydrogenase; presteady-state kinetics; pyrroloquinoline quinone; singular value decomposition; substrate-assisted catalysis