Syncatalytic inactivation of prolyl 4-hydroxylase by anthracyclines

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Abstract
The anthracyclines doxorubicin and daunorubicin were found to act as irreversible inhibitors of prolyl 4-hydroxylase. The reaction frate for enzyme from both chick and human origin was first order, the concentration of inhibitor giving 50% inhibition being 60 .mu.M for both compounds after 1 h. The effect was dependent on the presence of iron ions in the reaction mixture. Inactivation could be prevented by addition of high concentration of ascrobate, but not 2-oxoglutarate, before the inactivation period. The same results were obtained with competitive analogues of these cosubstrates. Lysyl hydroxylase from chick embryos was also susceptible to inactivation. Its activity was decreased by 50% after incubation for 1 h with a 150 .mu.M concentration of the inhibitors. When chick-embryo prolyl 4-hydroxylase was incubated with [14-14C]doxorubicin, both enzyme subunits were radioactively labelled, about 70% of the total radioactivity being found in the .alpha.-subunit. Since the anthracyclines are known to undergo a redox reaction generating semiquinone radicals with Fe3+ only, the results suggest that the enzyme-bound iron ion is oxidized to a tervalent intermediate in uncoupled reaction cycles. The data also suggest that both enzyme subunits contribute to the catalytic site of prolyl 4-hydroxylase.

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