Question

A human carbonic anhydrase mutant H64A binds well to Zn, which binds well to the substrate water. However, the k_cat is much lower than that of the wildtype, particularly at low pH. What might explain this?

Answer 1

Ans. To bind Zn²⁺ (or, Zn) the binding site shall preferably has negatively charged residues like aspartic acid (pKc = 3.71) and glutamic acid (pKc = 4.15). All the side chains (100 % deprotonation) of both residues would be negatively charged at pH around 6.0 and above. Since Zn²⁺ is used as cofactor, the better is the binding of cofactor to the enzyme, better would be catalysis.

# Given “Kcat is much lower than that of wild type, particularly as low pH”.

Kcat is a measure of number of substrates catalyzed per unit time by unit amount of enzyme. Lowering of Kcat means that the rate of catalysis (V_max) has been lowered.

# The lowering of Kcat of mutated enzyme is possible due to change on one or more acidic residues (Aspartic acid, glutamic acid) into basic residues (Arginine, Lysine, Proline). Presence of positive charge on the side chain of basic residues would repel positively charged Zn²⁺ ions, thus prevents the binding of cofactor.

Arginine has pKc of 10.76, lysine has pKc of 10.67 whereas proline has pKc of 6.04.

The side chain of arginine and lysine bear positive charge at a pH units lower than their pKc. That is, their side chains undergoes 100% protonation at pH 8 or lower – so, they are positively charged at physiological pH and below it.

However, proline would become 100% deprotonated and positively charged at pH 4.04 or lower. So, if the acidic residues are substituted by proline (basic residue), the Kcat of mutated protein would be drastically decreased at lower pH.

Conclusion: The mutated enzyme has one or more of its acidic residues at the Zn²⁺(cofactor) binding site replaced by proline.