How to Determine Enzyme Specificity

Enzyme specificity for different substrates is compared using catalytic efficiency, given by the ratio kcat/K_M.

• A higher kcat means the enzyme turns over substrate faster per active site per second.
• A lower K_M means higher affinity of the enzyme for the substrate.

Given kinetic data and catalytic efficiency

Fluorofumarate:

kcat/K_M = 2700 / 27 = 100 (sec⁻¹ μM⁻¹)

Fumarate:

kcat/K_M = 800 / 5 = 160 (sec⁻¹ μM⁻¹)

Chlorofumarate:

kcat/K_M = 20 / 111 ≈ 0.18 (sec⁻¹ μM⁻¹)

Thus, specificity (catalytic efficiency) decreases in the order:
Fumarate (160) > Fluorofumarate (100) > Chlorofumarate (0.18).

So the correct option is (C) Fumarate > Fluorofumarate > Chlorofumarate.

Explanation of Each Option

Option (A) Fluorofumarate > Fumarate > Chlorofumarate

This option incorrectly places fluorofumarate above fumarate in specificity. Since fumarate has a higher kcat/K_M (160) than fluorofumarate (100), fumarase is more specific for fumarate, not fluorofumarate, so this option is wrong.

Option (B) Chlorofumarate > Fluorofumarate > Fumarate

This option is incorrect because it places chlorofumarate as the most specific substrate, but chlorofumarate shows both high K_M (low affinity) and very low kcat (slow turnover). Its very low kcat/K_M (~0.18) makes it the least preferred substrate for fumarase.

Option (C) Fumarate > Fluorofumarate > Chlorofumarate

This option correctly follows the descending order of kcat/K_M: fumarate (160) > fluorofumarate (100) > chlorofumarate (~0.18). Fumarate is also the natural substrate of fumarase in the TCA cycle, and typically shows the highest specificity, consistent with this order.

Option (D) Fumarate > Chlorofumarate > Fluorofumarate

This option correctly places fumarate first but wrongly places chlorofumarate ahead of fluorofumarate. Since fluorofumarate has a much higher kcat/K_M than chlorofumarate, fumarase is more specific for fluorofumarate than for chlorofumarate, so this order is incorrect.