Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants

Franco, Ricardo; Tavares, Pedro; Mangravita, Arianna; Barber, Michael J.; Moura, Isabel; Ferreira, Glória C.

doi:10.1042/bj3560217

Cited by 14 publications

(16 citation statements)

References 20 publications

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“…This behavior requires that significant amounts of the EZnD IX complex exist in the steady state and therefore that ZnD IX disassociation is partially rate-limiting. This result is thus in agreement with previous transient kinetic studies using iron (16) and zinc (33,34) as metal ion substrates. This conclusion can be contrasted to previous work with the bovine enzyme (35) where steady-state inhibition studies using both product and alternative substrate demonstrated an ordered mechanism where iron bound first.…”

Section: Discussionsupporting

confidence: 83%

Metal Ion Selectivity and Substrate Inhibition in the Metal Ion Chelation Catalyzed by Human Ferrochelatase

Davidson

Chesters

Reid

2009

Journal of Biological Chemistry

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Protoporphyrin IX ferrochelatase (EC 4.99.1.1) catalyzes the terminal step in the heme biosynthetic pathway, the insertion of ferrous iron into protoporphyrin IX. Ferrochelatase shows specificity, in vitro, for multiple metal ion substrates and exhibits substrate inhibition in the case of zinc, copper, cobalt, and nickel. Zinc is the most biologically significant of these; when iron is depleted, zinc porphyrins are formed physiologically. Examining the k cat /K m app ratios for zinc and iron reveals that, in vitro, zinc is the preferred substrate at all concentrations of porphyrin. This is not the observed biological specificity, where zinc porphyrins are abnormal; these data argue for the existence of a specific iron delivery mechanism in vivo. We demonstrate that zinc acts as an uncompetitive substrate inhibitor, suggesting that ferrochelatase acts via an ordered pathway. Steady-state characterization demonstrates that the apparent k cat depends on zinc and shows substrate inhibition. Although porphyrin substrate is not inhibitory, zinc inhibition is enhanced by increasing porphyrin concentration. This indicates that zinc inhibits by binding to an enzyme-product complex (EZnD IX ) and is likely to be the second substrate in an ordered mechanism. Our analysis shows that substrate inhibition by zinc is not a mechanism that can promote specificity for iron over zinc, but is instead one that will reduce the production of all metalloporphyrins in the presence of high concentrations of zinc.Ferrochelatase (EC 4.99.1.1), the final enzyme of heme biosynthesis, catalyzes the insertion of ferrous iron into protoporphyrin IX (1); this reaction is commonly proposed to involve a distorted porphyrin intermediate (1,2). In vitro, ferrochelatase has a broad metal ion specificity with insertion of Zn 2ϩ , Co 2ϩ , and Cu 2ϩ having also been observed (1, 3). Metal ion specificity is species-dependent; the ferrochelatase from Bacillus subtilis accepts Cu 2ϩ but not Co 2ϩ as a substrate (4), in contrast to the widely reported specificity of most other ferrochelatases. Interestingly, it has recently been demonstrated that the poor activity toward Cu 2ϩ as a substrate, in the case of the murine and yeast ferrochelatases at least, arises from substantial substrate inhibition (3). Of the competing metal ions, Zn 2ϩ is perhaps the most biologically significant; under conditions of iron depletion or lead poisoning, zinc porphyrins can be formed physiologically (5), and the presence of zinc porphyrins in human blood can be used as a diagnostic test for lead poisoning (6). It has been suggested that the metal ion specificity of ferrochelatase is determined by the extent of porphyrin distortion in the active site of the enzyme (2). More recent crystallography has shown that some metal ion inhibitors are inserted into porphyrins, and the inhibition therefore arises from reduced product release (7).Crystal structures of free enzyme (8, 9), as well as enzyme with metal (10, 11), inhibitors (12, 13), porphyrin substrate (14), and a range of po...

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Section: Discussionsupporting

confidence: 83%

Metal Ion Selectivity and Substrate Inhibition in the Metal Ion Chelation Catalyzed by Human Ferrochelatase

Davidson

Chesters

Reid

2009

Journal of Biological Chemistry

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“…In the wild-type model, the N-terminal loop residues Gln 248 and Ser 249 were buried deeply inside the active site cavity and in very close proximity (about 4 Å) to His 209 and Glu 289 , which were proposed to be essential for catalysis (31)(32)(33)(34). Lys 250 was solvent-exposed, with the side chain projecting to the protein exterior and in close contact with the Gln 260 residue.…”

Section: Purification and Steady-state Kinetic Analysis Of Wild-type mentioning

confidence: 99%

Probing the Active Site Loop Motif of Murine Ferrochelatase by Random Mutagenesis

Shi

Ferreira

2004

Journal of Biological Chemistry

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Ferrochelatase catalyzes the terminal step of the heme biosynthetic pathway by inserting ferrous iron into protoporphyrin IX. A conserved loop motif was shown to form part of the active site and contact the bound porphyrin by molecular dynamics calculations and structural analysis. We applied a random mutagenesis approach and steady-state kinetic analysis to assess the role of the loop motif in murine ferrochelatase function, particularly with respect to porphyrin interaction.

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“…1, 2,3), but there are very few reports of transient kinetics (3,27). Most functional analysis and assessment of the significance of mutated residues has relied on steady-state information.…”

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confidence: 99%

Direct Measurement of Metal Ion Chelation in the Active Site of Human Ferrochelatase

et al. 2007

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The final step in heme biosynthesis, insertion of ferrous iron into protoporphyrin IX, is catalyzed by protoporphyrin IX ferrochelatase (E.C. 4.99.1.1). It is demonstrated that the pre-steady state human ferrochelatase (R115L) shows 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 ca. 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, converting 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 around 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 seven fold drop in k cat and three fold drop 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. Keywords porphyrin biosynthesis; ferrochelatase; transient kineticsThe final step in heme biosynthesis, insertion of ferrous iron into protoporphyrin IX, is catalyzed by protoporphyrin IX ferrochelatase (protoheme ferro-lyase, E.C. 4.99.1.1, the human enzyme is hereafter referred to as ferrochelatase). The mechanisms of biological iron chelation have proved to be of great interest with a range of studies examining reaction kinetics (1-3), spectroscopy of bound intermediates (4-6), sensitivity of the reaction to structural variation (1,3), and behavior of the enzyme analogues, including antibodies (4,7-9) and both DNA and RNA (10-12) that also catalyze metal ion insertion into porphyrins. Additionally crystal structures of free enzyme (13,14), as well as with bound metal substrate , bound porphyrin substrate (15) and a tight-binding competitive inhibitor (16) should support confident interpretation of the link between structure and function in this system. In fact, controversy remains over the role of individual residues in the reaction mechanism; compare for example the suggested metal ion binding site of Sellers et al. (1) with that proposed by Gora et al. † Funded by the BBSRC (UK).

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Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants

Cited by 14 publications

References 20 publications

Metal Ion Selectivity and Substrate Inhibition in the Metal Ion Chelation Catalyzed by Human Ferrochelatase

Metal Ion Selectivity and Substrate Inhibition in the Metal Ion Chelation Catalyzed by Human Ferrochelatase

Probing the Active Site Loop Motif of Murine Ferrochelatase by Random Mutagenesis

Direct Measurement of Metal Ion Chelation in the Active Site of Human Ferrochelatase

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