Dear colleagues, I appreciate your help in interpreting the pKa shift. Based on all the suggestions, I have several new leads to be able to come up with a tentative mechanism. I am summarizing the discussion below –
The pKa of a catalytically critical aspartic acid has increased to 6.44. It is hydrogen bonded (2.8 Angstroms) to a water molecule that is supposed to donate a proton during the catalysis. The pKa of the amino acid was estimated by the PropKa server (http://propka.ki.ku.dk/) using the co-ordinates of the crystal structure. Theoretically, pure water (assuming 55.5M) has only 1 proton in 5.55 x 10^ -6 molecules (correct me if I am wrong here). Or simply, water is not protonated at pH 7.0. Therefore, under physiological conditions, it is almost impossible for water to act as a nucleophile, acid or base by itself. An acid/base catalyst or a metal ion is usually employed by enzymes to activate water for catalysis. In this particular case, a solvent derived proton has been speculated to complete the reaction. An aspartic acid hydrogen bonded to this candidate solvent molecule seems a good choice for protonating the water molecule. There are no other residues or solvent molecules bonded to this potential proton donor water molecule. At pH 7.4, a carboxyl with a pKa of 6.4 will be 90% ionised (deprotonated) and 10% protonated. In a mechanism where the carboxylic acid donates a proton to the water which in turn donates a proton during catalysis, then elevating the pKa to 6.4 would be reasonable as the carboxyl will need to be protonated at some point in the cycle to do the donating. Raising the pKa of aspartic acid would allow a larger fraction of it to be in its protonated state at physiologically relevant pH values, although it would reduce the intrinsic effectiveness of Asp as a general acid. There should be a significant thermodynamic and kinetic advantage in having Asp participate directly in a general acid catalyzed reaction, rather than through a water molecule. Alternatively, the higher the pKa, the higher the nucleophilicity of the residue/group (higher SN2 reactivity or affinity with electrophiles, like H+, perhaps the substrate of your enzyme..?, etc). Suggested literature and tools 1. Some papers by Nick Pace's group: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2708032/?tool=pubmed http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2679426/?tool=pubmed http://www.sciencedirect.com/science/article/pii/S002228360600934X 2. Pace, C. et al. Protein Ionizable Groups: pK values and Their Contribution to Protein Stability and Solubility. J. Biol Chem. 284, 13285-13289 (May 15, 2009) 3. Harris TK & Turner GJ Structural basis of pertubed pKa values of catalytic groups in enzyme active sites IUBMB life, 53 85-98 (Feb 2002) 4. Papers of John A. Gerlt, who did a lot on protonabtraction reactions. 5. http://www.jinkai.org/AAD_history.html 6. THEMATICS for pKa calculations. Avaliable at http://www.northeastern.edu/org/wp/ 7. Tools to predict protein ionization http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2578799/ 8. Fap1_BBRC.pdf Apologies if I missed out on any reference or critical point/s raised in the discussion. Thank you. Deepak Oswal
