title: Evidence for an elevated aspartate pK(a) in the active site of human aromatase.
abstract: 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 Asp(309) in substrate binding and catalysis. These studies predicted an elevated pK(a) for Asp(309) and proposed that protonation of this residue was required for function. In this study, UV-visible 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, whereas, 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 Asp(309) with a parallel set of spectroscopic studies using exemestane and anastrozole. Because exemestane interacts with Asp(309) 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 pK(a) of 8.2 observed for androstenedione binding to the side chain of Asp(309). To our knowledge, this work represents the first experimental assignment of a pK(a) value to a residue in a cytochrome P450. This value is in agreement with theoretical calculations (7.7-8.1) despite the reliance of the computational methods on the conformational snapshots provided by crystal structures.
authors.affiliation: From the Department of Life Sciences and Systems Biology, University of Torino, via Accademia Albertina 13, 10123 Torino, Italy.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Di Nardo
authors.affiliation: From the Department of Life Sciences and Systems Biology, University of Torino, via Accademia Albertina 13, 10123 Torino, Italy.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Breitner
authors.affiliation: From the Department of Life Sciences and Systems Biology, University of Torino, via Accademia Albertina 13, 10123 Torino, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Bandino
authors.affiliation: the Department of Pharmacology, SUNY Upstate Medical University, Syracuse, New York 13210, and.
authors.forename: Debashis
authors.initials: D
authors.lastname: Ghosh
authors.affiliation: the Department of Physiology and Biophysics and the Massey Cancer Center, Virginia Commonwealth University School of Medicine, Richmond, Virginia 23219.
authors.forename: Gareth K
authors.initials: GK
authors.lastname: Jennings
authors.affiliation: the Department of Physiology and Biophysics and the Massey Cancer Center, Virginia Commonwealth University School of Medicine, Richmond, Virginia 23219 jchackett@vcu.edu.
authors.forename: John C
authors.initials: JC
authors.lastname: Hackett
authors.affiliation: From the Department of Life Sciences and Systems Biology, University of Torino, via Accademia Albertina 13, 10123 Torino, Italy, gianfranco.gilardi@unito.it.
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Gilardi
mesh_terms: D000728:Androgens / Q000737:chemistry / Q000378:metabolism; D001141:Aromatase / Q000737:chemistry* / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004967:Estrogens / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D009154:Mutation; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman
chemical_list: D000728:Androgens; D004967:Estrogens; D001224:Aspartic Acid; D001141:Aromatase; C561410:CYP19A1 protein, human
keywords: Aromatase; Aspartate (Aspartic Acid); Cytochrome P450; Enzyme Catalysis; Estrogen; Heme; Steroid; pKa