Year: 1984
Type: article
Abstract: 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 ( K , = 0.5 PM).The Michaelis constants for iron and zinc were 0.35 and 0.08 PM, respectively, and the inhibitory constants for competitive incorporation of iron and zinc were KIFelZa = 0.12 PM and KIZnFe = 0.58 p ~.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.The terminal step in heme biosynthesis, the insertion of ferrous iron in the porphyrin ring, is catalyzed by the enzyme ferrochelatase (EC 4.99.1.1).This membrane-bound enzyme is associated with the inner mitochondrial membrane in eukaryotic cells (1, 2).It has been purified to homogeneity from rat liver (3) and bovine liver (4) mitochondria.The membrane-bound and the purified enzymes exhibit a broad in vitro specificity toward substrates as different divalent metal ions, e.g.Fe2+, Zn2+, Co2+, or Ni", are enzymatically incorporated into different dicarboxylic porphyrins (1, 4-7).Extensive enzymatic studies of the crude enzyme or of its detergentsolubilized form from various origins have shown a random bi-bi mechanism of reaction of the enzyme (6-8), and a detailed study of the competition between iron and zinc * This work was supported hy United States Public Health Service Grants AM21682 and AM29742.The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.$ Performed this work during the French Service of Cooperation wit,h agreement of the French Ministry of Defense and Ministry of Foreign Relations.
Source: Journal of Biological Chemistry
Authors: J M Camadro, N G Ibraham, R D Levere
Institution: New York Medical College
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W1501090807