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Direct measurement of metal ion chelation in the active site of human ferrochelatase






Biochemistry 46(27): 8121-8127

Direct measurement of metal ion chelation in the active site of human ferrochelatase

The final step in heme biosynthesis, insertion of ferrous iron into protoporphyrin IX, is catalyzed by protoporphyrin IX ferrochelatase (EC 4.99.1.1). We demonstrate that pre-steady state human ferrochelatase (R115L) exhibits a stoichiometric burst of product formation and substrate consumption, consistent with a rate-determining step following metal ion chelation. Detailed analysis shows that chelation requires at least two steps, rapid binding followed by a slower (k approximate to 1 s(-1)) irreversible step, provisionally assigned to metal ion chelation. Comparison with steady state data reveals that the rate-determining step in the overall reaction, conversion of free porphyrin to free metalloporphyrin, occurs after chelation and is most probably product release. We have measured rate constants for significant steps on the enzyme and demonstrate that metal ion chelation, with a rate constant of 0.96 s(-1), is similar to 10 times faster than the rate-determining step in the steady state (k(cat) = 0.1 s(-1)). The effect of an additional E343D mutation is apparent at multiple stages in the reaction cycle with a 7-fold decrease in k(cat) and a 3-fold decrease in k(chel). This conservative mutation primarily affects events occurring after metal ion chelation. Further evaluation of structure-function data on site-directed mutants will therefore require both steady state and pre-steady state approaches.


Accession: 015516736

PMID: 17566985

DOI: 10.1021/bi602418e



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