Table 1: Summary table of pKa shifts in positions 199 and 222 presumably involved in catalysis, specified by species (HSA, BSA, and RabSA)
This activation seems to be related to the presence of basic residues that are in contact with the mentioned residues. We observed that position Lys 199 of HSA is abnormally acidic (average pKa among the conformers is ~7.51, see Supplementary Table 2 information about conformers used) while Arg 222 has a pKa ~9.49, also abnormally acid for an Arg residue. When we analyzed equivalent positions in BSA, we found that Arg 198 has a pKa ~9.24 (equivalent to Lys 199 of HSA). However, Lys 221 (equivalent to Arg 222 of HSA) shows a pKa ~7.27. As reported previously12,14, an acidic residue acting as a nucleophile is required to sustain the aldol condensation of acetone with p-formylbenzonitrile. This acid residue is postulated to be the Lys 199 in HSA and Lys 221 in BSA. In this sense, the “active site” is inverted in these two seroalbumins. As we discussed early, these positions are well conserved due to the observed substitutions of Lys and Arg, residues with positive charges that can be normally interchanged during evolution. However, physicochemical constraints related to the containing cavity introduced pKa shifts, although inverted, to support the promiscuous reaction. More interestingly, equivalent positions to Lys 199 and Arg 222 in HSA are occupied by two Arg in RabSA. The analysis of their pKa indicated that the amino-terminal Arg (Arg 199) showed an abnormal basic shift with a pKa ~16 while the carboxy-terminal Arg (Arg 222) has a pKa ~8. However, according to the experimental results shown in Figure 1, an acid Arg and a basic Arg sustain the reaction with much less efficiency than HSA and BSA, a fact which could explain taking into account the lesser nucleophilicity of arginine compared with lysine. Unfortunately, the other serum albumins used, PSA and RSA, lack a known crystallographic structure and pka shift estimation is not possible.