Enolase is an enzyme that catalyzes one reaction in glycolysis in all organisms that carry out this process. The amino acid sequence of enolase is similar but not identical in the organisms. Researchers purified enolase from Saccharomyces cerevisiae, a single-celled eukaryotic yeast that grows best at 37°C, and from Chloroflexus aurantiacus, a bacterium that grows best at the much higher temperature of 55°C. The researchers compared the activity of purified enolase from the two organisms by measuring the rate of the reaction in the presence of varying concentrations of substrate and a constant amount of each enzyme at both 37°C and 55°C.

(a) Depending on the organism, the optimal pH for enolase to catalyze its reaction is between 6.5 and 8.0. Describe how a pH below or above this range is likely to affect enolase and its catalytic ability.

(b) Identify the appropriate negative control the researchers most likely used when measuring the reaction rate in the presence of each organism’s enolase.

(c) The researchers predict that for any particular concentration of substrate, the C. aurantiacus enolase-catalyzed reaction is more rapid at 55°C than at 37°C. Provide reasoning to justify the researchers’ prediction.

a) The response indicates that a pH below or above this range will most likely cause enolase to denature/change its shape and be less efficient or unable to catalyze the reaction.

b)The response indicates that the appropriate negative control is to measure the reaction rate (at the varying substrate concentrations) without any enzyme present.

c)The response indicated that the enolase has a more stable/functional/correct/normal protein structure at the higher temperature of 55°C than at 37°C because the enzyme is from an organism that is adapted to growth at 55°C.

Explanation:

Enolase catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate during both glycolysis and gluconeogenesis.In bacteria, enolases are highly conserved enzymes and commonly exist as homodimers.

The temperature optimum for enolase catalysis was 80°C, close to the measured thermal stability of the protein which was determined to be 75°C, while the pH optimum for enzyme activity was 6.5. The specific activities of purified enolase determined at 25 and 80°C were 147 and 300 U mg−1 of protein, respectively. Km values for the 2-phosphoglycerate/phosphoenolpyruvate reaction determined at 25 and 80°C were 0.16 and 0.03 mM, respectively. The Km values for Mg2+ binding at these temperatures were 2.5 and 1.9 mM, respectively.

Enolase-1 from Chloroflexus aurantiacus (EnoCa), a thermophilic green non-sulfur bacterium that grows photosynthetically under anaerobic conditions. The biochemical and structural properties of enolase from C. aurantiacus are consistent with this being thermally adapted.