[kingbanks]

Hey guys! First time poster here, stuck on a (not for credit) homework problem in pharm school - how do I assign pKa values to Histidine residues 12 and 119 based on this titration curve:

http://imgur.com/ZOkixwT

and this mechanism:

http://proteopedia.org/wiki/images/thumb/d/d6/MechIII.png/400px-MechIII.png

This website identifies which residue has which pKa but doesn't do a great job of explaining it:

https://mcdb-webarchive.mcdb.ucsb.edu/sears/biochemistry/tw-enz/rnasea/rnasea-overview.htm

Basically, why does His 12 have the lower pKa despite it being the more basic residue at the beginning and end of the rxn?

Any help is appreciated! Thanks in advance

[Babcock_Hall]

Can you give your thoughts?  My hint is that it is easier to study this reaction by separating it into halves.  A 2', 3' cyclic phosphodiester intermediate can be isolated, and one can study its reaction (which is the second half of the entire reaction) independently.
Kinetic and Equilibrium Studies of the Ribonuclease-Catalyzed Hydrolysis of Uridine 2', 3'Cyclic Phosphate"
Ernest J. del Rosariot and Gordon G. Hammes, Biochemistry 8 (1969).
EDT
I am pressed for time right now, but this is an interesting subject and I might be able to contribute more thoughts later.

[Babcock_Hall]

One really should define what is meant by "activity" before proceeding.  Is it k_cat or k_cat/K_m?  They often give different pH profiles, and the pK_a values of enzymatic groups often change when substrate binds.  It would also be helpful to whether the rate being measured is for one full catalytic turnover, or for the hydrolysis of the intermediate, the 2', 3' cyclic phosphodiester (this is the second half reaction).  My understanding is that one sees similar results for the substrate versus the intermediate (Hammes, 1969), but I am not certain.

However just taking the pH-rate profile that is given and assuming that we are talking about the second half-reaction, I would reason as follows:  The pK_a of 5.4 represents a group which must be deprotonated for the reaction to proceed (activity decreases as pH is lowered beyond that point).  This might be a base or a nucleophile (if we were studying a different enzyme), but for ribonuclease it is probably a base.  In the drawing of the second half-reaction, the base is His119.  The pK_a of 6.4 represents a group that must be protonated for the reaction to proceed (activity decreases as pH become greater than this).  This might be a general acid.  For the second half-reaction of ribonuclease, the general acid is His12.