Substrate recognition by prolyl 4-hydroxylase

The post-translational hydroxylation of proline is found in a growing number of proteins, but is most widely known in collagen, a structural protein in animals. The conversion of (2S)-proline (Pro) to (2 S,4R)-hydroxyproline (Hyp) stabilizes the triple-helical structure of collagen. Hydroxylation of proline residues is catalyzed by the enzyme prolyl 4-hydroxylase (P4H). P4H utilizes iron(II), molecular oxygen, and alpha-ketoglutarate as co-substrates to hydroxylate proline residues in a polypeptide substrate. This dissertation focuses on how P4H recognizes its substrates.;P4H catalyzes the hydroxylation of proline residues within Xaa-Pro-Gly sequences, where Xaa is any amino acid and Gly is glycine. The amino acids surrounding Pro influence the peptide structure and hydroxylation of Pro, but the conformation of Pro recognized by P4H was unknown. CHAPTER Two describes the use of proline analogues to determine that P4H prefers the proline Cgamma-endo ring pucker conformation. This study proposes a mechanism by which P4H avoids product inhibition. This information will facilitate future design of inhibitors of the enzyme.;The study of enzymes, including P4H, relies heavily on the ability to assay the activity of the enzyme quickly, easily, and accurately. Because P4H uses a number of co-substrates and the natural substrate, collagen, is so large, no efficient assay of catalytic turnover by prolyl hydroxylation had been developed. In CHAPTER THREE, we describe the design and use of an assay that is both continuous and direct by utilizing (2S,4S)-4-fluoroproline. This assay is demonstrated to be useful for both mechanistic studies and the screening of inhibitors.;P4H binds iron(II) in its active site via two histidines and one aspartate residue, termed a 2-His-l-carboxylate motif. This motif is common to many iron(II) dioxygenases similar to P4H. A class of related enzymes catalyzes halogenation reactions, rather than hydroxylations. These halogenases contain two histidine iron-binding residues, but lack the aspartate. Instead, a halide replaces the carboxylate. CHAPTER FOUR describes the conversion of P4H to mimic the halogenase active site and the analysis of the orientation of the iron-binding residues in P4H. These experiments demonstrate that the native P4H active site is resistant to alteration and critical for any catalytic activity.