Soluble quinoprotein (PQQ-containing) glucose dehydrogenase (sGDH,
ac.uk/iubmb/enzyme/EC1/1/99/35.html" title="Link to external resource: http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/1/99/35.html" target="_blank">EC 1.1.99.35) catalyzes the oxidation of β-
d-glucose to
d-glucono-δ-l
actone. 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 inter
action. To investigate this conclusion, stopped-flow kinetic experiments were carried out on the re
action 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 f
acilitated by binding of substrate to Int. Substituting the PQQ-
activating Ca by a Ba ion lowered all re
action rates but did not change the stimulation f
actor. In summary, the previous proposal that the cooperativity effect of sGDH is due to inter
action between its substrate-loaded subunits is incorrect; it is due to substrate-assisted catalysis of the enzyme.