Formation of enzyme-substrate complexes with protocollagen proline hydroxylase and large polypeptide substrates.

Cartilage from chick embryos was incubated with l4Cproline and puromycin in order to prepare puromycin peptides which were substrates for the synthesis of 14C-hydroxyproline with protocollagen proline hydroxylase. Stable enzymesubstrate complexes were recovered when the substrate was mixed with the enzyme and the mixtures were examined by gel filtration. Although ascorbate, ar-ketoglutarate, and ferrous iron are required for catalytic activity, formation of the enzymesubstrate complexes did not require addition of the cofactors or cosubstrates to dialyzed enzyme. Formation of the complexes was reduced but not prevented by EDTA in concentrations which inhibited catalytic activity. Under the conditions used, the enzyme in the isolated complexes was readily saturated with substrate, but it was difficult to saturate the substrate with enzyme. With high concentrations of enzyme reiative to substrate, the initial velocity for the reaction was greater than the initial velocity observed with the isolated enzyme-substrate complexes, suggesting that 1 molecule of polypeptide substrate could bind more than 1 molecule of enzyme. The enzyme-substrate complexes were in equilibrium with free enzyme and substrate, since addition of a competing polypeptide produced a dissociation of the initial complexes and a rapid inhibition of the hydroxylation of the substrate. Most of the substrate was still recovered in the enzymesubstrate complexes under conditions in which the over-all concentration of substrate was about 100 PM polypeptide. Since the enzyme appeared to be saturated with substrate under these conditions, the results suggested that the dissociation constant for the complexes expressed in terms of the molar concentration of substrate polypeptides was probably less than 100 PM. The afhnity of the enzyme for substrate decreased after partial hydroxylation of the substrate. After one-sixth of the available 14C-proline in the substrate was converted to 14C-hydroxyproline, it was no longer possible