Kinetic studies of human liver ferrochelatase. Role of endogenous metals.
Ferrochelatase activity in human liver has been extensively characterized in the mitochondrial fraction by kinetic study of the enzyme in initial velocity conditions. We found that human liver mitochondrial membranes contain large amounts of endogenous metals that are substrates for the enzyme, leading to a lack of linearity of the activity as function of protein concentration. This lack of linearity is mainly due to a high zinc-chelatase activity with endogenous zinc. Under optimal experimental conditions, the maximum velocity for iron incorporation was 8.7 nmol of protoheme/h/mg of protein, and the maximum velocity for zinc incorporation was 4.3 nmol of zinc-protoporphyrin/h/mg of protein. The Michaelis constant for protoporphyrin IX was (i) dependent on the amount of protein when the overall chelatase reactions were measured but (ii) independent of the amount of protein when only zinc-chelatase activity was measured (Km = 0.5 microM). The Michaelis constants for iron and zinc were 0.35 and 0.08 microM, respectively, and the inhibitory constants for competitive incorporation of iron and zinc were KIFe/Zn = 0.12 microM and KIZn/Fe = 0.58 microM. The affinity of the enzyme for zinc lowers the actual determination of ferrochelatase activity with iron as substrate. Furthermore, when measuring ferrochelatase (e.g. in liver biopsy), endogenous zinc content in the biological sample must be taken into account.
D015082:2,2'-Dipyridyl / Q000494:pharmacology; D000693:Anaerobiosis; D004492:Edetic Acid / Q000494:pharmacology; D005294:Ferrochelatase / Q000378:metabolism*; D006801:Humans; D007501:Iron / Q000494:pharmacology*; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000201:enzymology*; D011524:Protoporphyrins / Q000494:pharmacology; D013053:Spectrophotometry; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology*
D011524:Protoporphyrins; D015082:2,2'-Dipyridyl; D004492:Edetic Acid; C028025:protoporphyrin IX; D007501:Iron; D008190:Lyases; D005294:Ferrochelatase; D015032:Zinc