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Formation of Enzyme-Substrate Complexes with Protocollagen Proline Hydroxylase and Large Polypeptide Substrates

From the ¹ From the Departments of Medicine and Biochemistry, University of Pennsylvania, and the Philadelphia General Hospital, Philadelphia, Pennsylvania 19104

Cartilage from chick embryos was incubated with ¹⁴C-proline and puromycin in order to prepare puromycin peptides which were substrates for the synthesis of ¹⁴C-hydroxyproline with protocollagen proline hydroxylase. Stable enzyme-substrate complexes were recovered when the substrate was mixed with the enzyme and the mixtures were examined by gel filtration.

Although ascorbate, -ketoglutarate, and ferrous iron are required for catalytic activity, formation of the enzyme-substrate 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 relative 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 enzyme-substrate 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 affinity 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 to demonstrate enzyme-substrate complexes in the gel filtration column.

Previous kinetic measurements demonstrated that the enzyme has a higher affinity for large polypeptides than for small ones of the same structure, and that a single encounter between enzyme and a large polypeptide probably involves a large segment of the polypeptide. The present results indicate that multiple encounters between enzyme and substrate are necessary to complete the hydroxylation of polypeptide substrates with molecular weights of up to 100,000. Since the affinity of the enzyme for the substrate decreases after partial hydroxylation of the substrate, the introduction of the final few hydroxyl groups into large polypeptide substrates apparently does not proceed as readily as the introduction of the first few hydroxyl groups.

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