KINETIC PATTERNS OF PROTOCOLLAGEN HYDROXYLASE AND FURTHER STUDIES ON POLYPEPTIDE SUBSTRATE

The kinetics of the protocollagen hydroxylase reaction were examined by the technique of measuring the initial velocities as a function of the concentration of one reactant with a series of fixed concentrations of a second reactant, and with a constant concentration of the remaining reactants. From the data a rate equation was developed for the interaction of the enzyme with ascorbate, α-ketoglutarate, oxygen, and polypeptide substrate. The kinetic data were consistent with a scheme involving consecutive bimolecular additions of ascorbate and α-ketoglutarate followed by the random order addition of oxygen and polypeptide substrate, but other kinetic schemes were not excluded. Studies with a purified enzyme system indicated that α-ketoglutarate was not consumed in stoichiometric amounts in the reaction. Further studies on the polypeptide substrate supported earlier indications that the enzyme can hydroxylate polypeptides without triple helical structure. When proline-labeled [14C]protocollagen was boiled and quenched, the synthesis of [14C]-hydroxyproline was 74% of the control value. Also, the polypeptide (Gly-l-Ala-l-Pro)n which apparently does not have triple helical structure in solution was found to serve as a substrate for the synthesis of hydroxyproline by the enzyme. The Km value and maximum velocity for (Gly-l-Ala-l-Pro)n were slightly lower than for (l-Pro-Gly-l-Pro)n under the same conditions. © 1968.