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Ascorbate action on normal and mutant human lysyl hydroxylases from cultured dermal fibroblasts.

The mechanism of ascorbate action on lysyl hydroxylase (LH) was investigated using cultured dermal fibroblasts from a patient with hydroxylysine-deficient collagen disease and from appropriate control patients. Cells from this patient had moderately impaired LH and both parents had intermediate values. Prolyl hydroxylase (PH) was normal in these 3 cell lines and the ratio between LH and PH proved an effective genetic discriminant among cell lines from different individuals expressing varying enzyme activity. Ascorbate requirements for normal and mutant enzymes were defined and compared. Some specificity for ascorbate as the principal physiological reductant was found. Lysyl hydroxylase activity from controls was 89% of maximum when dithiothreitol alone was omitted whereas only 8% of activity was present when L-ascorbate alone was omitted. Control activity was fully reconstituted when isoascorbate was substituted for L-ascorbate, but activity was absent when dehydroascorbate was used. Enzyme activity in cultured cells from this patient and a control was enhanced proportionately by increasing ascorbate concentration. At concentrations of 0.5mM and above, saturation was evident and mutant enzyme activity was 17% of control. The apparent K m of both enzymes for ascorbate was identical (0.1 mM) while the apparent V max of mutant enzyme was reduced to 25% of control. Ascorbate did not alter thermostability of either mutant or control enzyme, and increasing ascorbate concentration did not overcome competitive inhibition by Cu 2+ of Fe 2+ -dependent enzyme activity. These studies demonstrated that this patient with type VI, Ehlers-Danlos syndrome had reduced skin collagen hydroxylysine caused by impaired lysyl hydroxylase, that this mutation was transmitted as an autosomal recessive trait and that the mutant enzyme had a reduced capacity for ascorbate. Ascorbate augmented lysyl hydroxylase activity by direct electron donation at a ferrous ion-containing site of this enzyme complex.