In the hexokinase–based glucose assay, which enzyme acts as the coupling step to allow NADPH-based detection?

In the hexokinase–based glucose assay, which enzyme acts as the coupling step to allow NADPH-based detection?

• A. Glucose dehydrogenase
• B. Glucose-6-phosphatase
• C. Glucose-6-phosphate dehydrogenase
• D. Peroxidase

Answer: (C)

In this assay, the signal comes from NADPH produced in a second, coupling reaction. After hexokinase converts glucose to glucose-6-phosphate, glucose-6-phosphate dehydrogenase oxidizes that product to 6-phosphogluconolactone and reduces NADP+ to NADPH. The formed NADPH is what you detect, giving the measurable signal for glucose concentration. Other enzymes don’t serve this coupling role: glucose dehydrogenase would directly oxidize glucose (not the hexokinase product path) and typically uses NAD(P)+ but isn’t the step coupling to the hexokinase reaction; glucose-6-phosphatase would remove the phosphate, undoing the intended step instead of generating NADPH; peroxidase is used in different assay formats to detect hydrogen peroxide, not to generate NADPH for this detection scheme.

In this assay, the signal comes from NADPH produced in a second, coupling reaction. After hexokinase converts glucose to glucose-6-phosphate, glucose-6-phosphate dehydrogenase oxidizes that product to 6-phosphogluconolactone and reduces NADP+ to NADPH. The formed NADPH is what you detect, giving the measurable signal for glucose concentration.

Other enzymes don’t serve this coupling role: glucose dehydrogenase would directly oxidize glucose (not the hexokinase product path) and typically uses NAD(P)+ but isn’t the step coupling to the hexokinase reaction; glucose-6-phosphatase would remove the phosphate, undoing the intended step instead of generating NADPH; peroxidase is used in different assay formats to detect hydrogen peroxide, not to generate NADPH for this detection scheme.