Question

Studies (kinetic, stereochemical, chemical modification, and site-specific mutagenesis experiments) on the enzyme triosephosphate isomerase have identified the B as Glu165 and HA as His95 (J. R. Knowles, Nature 350, 121 (1991). To aid your understanding, draw the active site as a pocket that extends into the enzyme and place the Flu and His on opposite sides of the pocket. Answer the following questions:

a. Drawing of the pocket:

b. How might the replacement of Glu with Asp affect the catalytic rate?

c. Discuss what form His would be in at a physiological pH of 7.2. Would that pH allow it to act as a general acid? Describe the conditions that allow it to act as a general acid. (hint: what reaction does a Ka refer to?)

Answer 1

Ans. b. The side chain of both Glu and Asp are acidic and bear 1 unit negative charge at physiological pH.

Therefore, Asp, being similar in size, shape (of side chain) and the same in charge of Glu, is most likely to retain catalytic activity unaffected.

Answer C. Histidine has a basic side chain. It accepts a proton as follow-   

His + H+ <------> His-H+

The pKa of the histidine side chain is 6.04.

Using Henderson-Hasselbalch equation for base-

  pH = pKa + log ([B] / [BH+])

7.2 = 6.04 + log ( [His] / [His-H+])

Or, [His] / [His-H+] = antilog (- 1.16) = 0.0692

Or, [His] = 0.0692 [His-H+]

Assuming total [H+] + [His-H+] = 1, the net positive charge on His residue is +0.0692.

Or, around 6.9 % of the total His residues would be positively charged whereas the rest would be neutral.

# Effect on catalysis: His has a cyclic side-chain whereas Glu linear side chain. Around 6.9% His residues are positively charged at pH 7.2 whereas all the Glu residues are negatively charged at the same pH. So, presence of positive charge on His as well as its inability of accepting a proton in (His-H+) state, is most likely to reduce the rate of catalysis at least by around 7% (even if the shape of the side chain is ignored.

# His as a general acid: To act as an acid, the His residues must be in protonated state (His-H+) so that it can donate an H+ like an acid.

To be predominantly in the protonated state (His-H+), the pH must be around 2 units lower than the pKa value of the base.

So, the desired pH = 4.04, at which almost all the His residues are in protonated (His-H+) state. And, His-H+ would act as an acid.

[You can try the HH equation to verify it].