Comparison between avian and human prolyl 4‐hydroxylases: Studies on the holomeric enzymes and their constituent subunits

Prolyl 4‐hydroxylase, a key enzyme in collagen biosynthesis, catalyzes the conversion of selected prolyl residues to trans ‐hydroxyproline in nascent or completed pro‐α chains of procollagen. The enzyme is a tetramer composed of two nonidentical subunits, designated α and β. To compare the enzyme and its subunits from different sources, the chick embryo and human placental prolyl 4‐hydroxylases were purified to homogeneity and their physicochemical and immunological properties were determined. Both enzymes were glycoproteins with estimated apparent molecular weights ranging between 400 and 600 kDa. Amino acid and carbohydrate analyses showed slight differences between the two holomeric enzymes, consistent with their deduced amino acid sequences from their respective cDNAs. Human placental prolyl 4‐hydroxylase contained more tightly bound iron than the chick embryo enzyme. Immunodiffusion of the human placental enzyme with antibodies raised against the purified chick embryo prolyl 4‐hydroxylase demonstrated partial identity, indicating different antigenic determinants in their tertiary structures. The enzymes could be separated by high‐resolution capillary electrophoresis, indicating differential charge densities for the native chick embryo and human placental proteins. Electrophoretic studies revealed that the human prolyl 4‐hydroxylase is a tetrameric enzyme containing two nonidentical subunits of about 64 and 62 kDa, in a ratio of approximately 1 to 2, designated α and β, respectively. In contrast, the chick embryo α and β subunit ratio was 1 to 1. Notably, the human α subunit was partially degraded when subjected to electrophoresis under denaturing conditions. Analogously, when the chick embryo enzyme was subjected to limited proteolysis, selective degradation of the α subunit was observed. Finally, only the α subunit was bound to Concanavalin A demonstrating that the α subunits of prolyl 4‐hydroxylase in both species were glycosylated. Using biochemical techniques, these results demonstrated that the 4‐ trans ‐hydroxy‐ L ‐proline residues in human placental collagens are synthesized by an enzyme whose primary structure and immunological properties differ from those of the previously well‐characterized chick embryo enzyme, consistent with their recently deduced primary structures from cDNA sequences.