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...
SUMMARY1. The absorption of glucose and fructose derived from sucrose has been studied using in vitro and in vivo loops of the rat jejunum.2. At low sucrose concentrations (1 and 10 mM) glucose appeared in the serosal compartment of the in vitro preparation at a faster rate than fructose, but at high sucrose concentrations (50 and 100 mm) the rates of serosal transfer of the two sugars were similar. Glucose and fructose appeared in the mucosal compartment, with the rate of fructose appearance exceeding that of glucose, at all the sucrose concentrations studied.3. Phlorizin (5 x IO-' M)-added to the mucosal medium of the in vitro preparation abolished the serosal transfer of glucose derived from 50 mM sucrose, and reduced that of fructose by 75 %.4. In the absence of sodium ions, the in vitro preparation failed to transfer glucose and fructose derived from 50 mM sucrose, into the serosal compartment.5. Glucose was actively accumulated in the whole gut wall of the in vivo preparation to concentrations higher than those in the plasma at 50 and 100 mM, but not at 10 mm sucrose concentrations. Fructose was also actively accumulated to about half the extent of glucose, but reached tissue concentrations greater than those in the plasma, at each sucrose concentration.6. The whole wall concentrations of glucose and fructose derived from sucrose added to the lumen continued to rise when the blood supply to the in vivo preparation was terminated.7. No increase in the in vivo whole wall concentrations of glucose and fructose were detected when sucrose was added to the lumen together with concentrations of glucose sufficient to saturate the monosaccharide transport systems.8. The results favour the view that disaccharide hydrolysis and resulting hexose transfer are sequential, separate events.
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