No abstract

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...

Enzymatischer Thermoschalter: Die bakteriellen 4‐Methylidenimidazol‐5‐on‐abhängigen Enzyme AdmH und EncP zeigen bemerkenswerte thermische Bifunktionalität: Bei niedriger Temperatur agieren sie als Mutasen (blauer Graph) und bei höheren Temperaturen hauptsächlich als Lyasen (roter Graph). Dieser temperaturabhängige Wechsel der Enzymklasse erklärt auch, warum diese ähnlichen Enzyme unterschiedliche katalytische Funktionen im sekundären Metabolismus wahrnehmen können.