Benchmarking pKa prediction methods for Lys115 in acetoacetate decarboxylase.

Three different pKa prediction methods were used to calculate the pKa of Lys115 in acetoacetate decarboxylase (AADase): the empirical method PROPKA, the multiconformation continuum electrostatics (MCCE) method, and the molecular dynamics/thermodynamic integration (MD/TI) method with implicit solvent. As expected, accurate pKa prediction of Lys115 depends on the protonation patterns of other ionizable groups, especially the nearby Glu76. However, since the prediction methods do not explicitly sample the protonation patterns of nearby residues, this must be done manually. When Glu76 is deprotonated, all three methods give an incorrect pKa value for Lys115. If protonated, Glu76 is used in an MD/TI calculation, the pKa of Lys115 is predicted to be 5.3, which agrees well with the experimental value of 5.9. This result agrees with previous site-directed mutagenesis studies, where the mutation of Glu76 (negative charge when deprotonated) to Gln (neutral) causes no change in Km, suggesting that Glu76 has no effect on the pKa shift of Lys115. Thus, we postulate that the pKa of Glu76 is also shifted so that Glu76 is protonated (neutral) in AADase. Graphical abstract Simulated abundances of protonated species as pH is varied.