Substrate specificity of the monooxygenase activity of hemoglobin.
Hemoglobin has been characterized as a monooxygenase-like catalyst of aniline hydroxylation both in reconstituted systems [ Mieyal et al. J. Biol. Chem. 251:3436-3441 (1976)] and in intact erythrocytes [ Blisard and Mieyal , J. Biol. Chem. 254:5104-5110 (1979)]. In this report, the monooxygenase activity of isolated hemoglobin (Hb) in the reconstituted system, which includes NADPH and cytochrome P-450 reductase, was shown to include N- and O-demethylation reactions besides p-hydroxylation, and to extend to other typical cytochrome P-450 substrates such as benzphetamine and p-nitroanisole. Some substrates were tested also with intact erythrocytes. Those which were metabolized displayed relative activities qualitatively similar to the pattern with isolated Hb. With isolated hemoglobin, complete kinetic analysis was carried out for 10 different reactions. The Km and Vmax values varied broadly, so that the efficiencies of the reactions (Vmax/Km) encompassed a range greater than 40,000. The most efficient reaction was O-demethylation of p-nitroanisole; the highest Vmax was observed for the O-demethylation of p-anisidine. The efficiencies (Vmax/Km) for a series of anisole derivatives (p-NH2,p-OH, p-H,p-NO2) was found to be quite sensitive to the electron-withdrawing effect of the p-substituent, i.e. a linear Hammett sigma rho relationship (log Vmax/Km versus sigma) was observed (p = 1.43). Metabolism of N-methylaniline by hemoglobin displayed distinct regioselectivity , with N-demethylation being favored over p-hydroxylation. Separate Km and Vmax values were observed for these two reactions of the single substrate, suggesting that distinct ternary O2-Hb-substrate complexes mediate the two reactions. In separate experiments, the various substrates were tested for their ability to accelerate autooxidation of HbO2 in the absence of NADPH and reductase. Aniline and its derivatives induced autooxidation with a concentration dependence matching their Km values for the corresponding hydroxylation reactions with the complete catalytic system. With the exception of p-hydroxyanisole, none of the other substrates accelerated autooxidation of HbO2. Hence this phenomenon cannot be an indicator of potential monooxygenase reactivity with hemoglobin. The broad and regioselective activities observed for hemoglobin resemble the characteristics of the authentic monooxygenase enzyme cytochrome P-450.[1]References
- Substrate specificity of the monooxygenase activity of hemoglobin. Starke, D.W., Blisard, K.S., Mieyal, J.J. Mol. Pharmacol. (1984) [Pubmed]
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