To design a new method to measure enzyme activity using zero-order kinetics, which substrate concentration should you initially determine experimentally?

• A. Substrate concentration b
• B. Substrate concentration a
• C. Substrate concentration c
• D. Substrate concentration d

Answer: (A)

In zero-order kinetics, the rate is independent of substrate concentration once the enzyme is saturated. That means at high enough substrate levels, all enzyme active sites are occupied, and the reaction rate reflects only how much enzyme is present, not how much substrate there is. To design a method around this, you first need to identify the substrate concentration that drives the system into saturation. Do this by measuring initial rates across a range of substrate levels, plotting rate versus substrate, and finding the plateau where increasing substrate no longer increases the rate. Use a substrate concentration in that plateau for the assay, ensuring zero-order behavior with respect to substrate. Substrates values in the rising portion of the curve would not give zero-order kinetics, so they’re not suitable for this approach.