Evidence for an elevated aspartate pKa in the active site of human aromatase

Aromatase (CYP19A1), the enzyme that converts androgens to estrogens, is of significant mechanistic and therapeutic interest. Crystal structures and computational studies of this enzyme shed light on the critical role of D309 in substrate binding and catalysis. These studies predicted an elevated pKa for D309 and proposed that protonation of this residue was required for function. In this study, UV-vis absorption, circular dichroism, resonance Raman spectroscopy, and enzyme kinetics were used to study the impact of pH on aromatase structure and androstenedione binding. Spectroscopic studies demonstrate that androstenedione binding is pH-dependent, while in contrast, the D309N mutant retains its ability to bind to androstenedione across the entire pH range studied. Neither pH nor mutation perturbed the secondary structure or heme environment. The origin of the observed pH dependence was further narrowed to the protonation equilibria of D309 with a parallel set of spectroscopic studies using exemestane and anastrozole. Since exemestane interacts with D309 based on its co-crystal structure with the enzyme, its binding is pH-dependent. Aromatase binding to anastrozole is pH-independent, consistent with the hypothesis that this ligand exploits a distinct set of interactions in the active site. In summary, we assign the apparent pKa of 8.2 observed for androstenedione binding to the side chain of D309. To our knowledge, this work represents the first experimental assignment of a pKa value to a residue in a cytochrome P450. This value is in agreement with theoretical calculations (7.7-8.1) despite the computational methods reliance on the conformational snapshots provided by crystal structures.