You are investigating a novel metabolic pathway involving the conversion of A to B by enzyme X. When ATP is depleted, the conversion rate of A to B decreases dramatically. Which experimental approaches would help elucidate the role of ATP in this reaction?
A. Measure the reaction rate of enzyme X in the presence of a non-hydrolyzable ATP analog.
B. Measure the binding affinity of A to enzyme X with and without ATP.
C. Assess whether ATP hydrolysis is coupled to the conversion of A to B by measuring inorganic phosphate release.
D. Increase ATP concentration and determine whether this alters the enzyme’s Km for A.