Studies on the lysyl hydroxylase reaction. I. Initial velocity kinetics and related aspects

Abstract

The kinetics of the lysyl hydroxylase (peptidyllysine, 2-oxoglutarate:oxygen 5-oxidoreductase, EC 1.14.11.4) reaction were studied using enzyme from chick embryos by varying the concentration of one substrate in the presence of different fixed concentrations of the second substrate, while the concentrations of the other substrates were held constant. Intersecting lines were obtained in double-reciprocal plots for all possible pairs involving Fe 2+, α-ketoglutarate, O 2 and the peptide substrate, whereas parallel lines were obtained for pairs comprising ascorbate and each of the other substrates. The pair composed of Fe 2+ and α-ketoglutarate gave an asymmetrical initial velocity pattern, indicating binding of these two reactants in this order, that of Fe 2+ being at thermodynamic equilibrium. The initial velocity patterns are identical with those reported for prolyl 4-hydroxylase, and the apparent K m and K d values calculated from these data are also very similar. The largest difference was found in K m and K d for α-ketoglutarate, which were about 4 times the corresponding values for prolyl 4-hydroxylase. Ascorbate was found to be a quite specific requirement for lysyl hydroxylase, but the enzyme catalyzed its reaction for a short time at a high rate in the complete absence of this vitamin, suggesting that the reaction with ascorbate does not occur during each catalytic cycle. Lysyl hydroxylase catalyzed an uncoupled decarboxylation of α-ketoglutarate in the absence of the peptide substrate, the rate being about 4% of that observed in the presence of a saturating concentration of the peptide substrate. This uncoupled decarboxylation required the same cosubstrates as the complete reaction.