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Product release rather than chelation determines metal specificity for ferrochelatase.

ABSTRACT: Ferrochelatase (protoheme ferrolyase, E.C. 4.99.1.1) is the terminal enzyme in heme biosynthesis and catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme IX (heme). Within the past two years, X-ray crystallographic data obtained with human ferrochelatase have clearly shown that significant structural changes occur during catalysis that are predicted to facilitate metal insertion and product release. One unanswered question about ferrochelatase involves defining the mechanism whereby some metals, such as divalent Fe, Co, Ni, and Zn, can be used by the enzyme in vitro to produce the corresponding metalloporphyrins, while other metals, such as divalent Mn, Hg, Cd, or Pb, are inhibitors of the enzyme. Through the use of high-resolution X-ray crystallography along with characterization of metal species via their anomalous diffraction, the identity and position of Hg, Cd, Ni, or Mn in the center of enzyme-bound porphyrin macrocycle were determined. When Pb, Hg, Cd, or Ni was present in the macrocycle, the conserved pi helix was in the extended, partially unwound "product release" state. Interestingly, in the structure of ferrochelatase with Mn-porphyrin bound, the pi helix is not extended or unwound and is in the "substrate-bound" conformation. These findings show that at least in the cases of Mn, Pb, Cd, and Hg, metal "inhibition" of ferrochelatase is not due to the inability of the enzyme to insert the metal into the macrocycle or by binding to a second metal binding site as has been previously proposed. Rather, inhibition occurs after metal insertion and results from poor or diminished product release. Possible explanations for the lack of product release are proposed herein.

SUBMITTER: Medlock AE

PROVIDER: S-EPMC2771925 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Product release rather than chelation determines metal specificity for ferrochelatase.

Medlock Amy E AE Carter Michael M Dailey Tamara A TA Dailey Harry A HA Lanzilotta William N WN

Journal of molecular biology 20090822 2

Ferrochelatase (protoheme ferrolyase, E.C. 4.99.1.1) is the terminal enzyme in heme biosynthesis and catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme IX (heme). Within the past two years, X-ray crystallographic data obtained with human ferrochelatase have clearly shown that significant structural changes occur during catalysis that are predicted to facilitate metal insertion and product release. One unanswered question about ferrochelatase involves defining the me ...[more]

PMID: 19703464

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Project description:BackgroundDextransucrases are extracellular enzymes, which catalyze the formation of α-1→6-linked glucose polymers from sucrose. These enzymes are exclusively expressed by lactic acid bacteria, which commonly acidify the extracellular environment due to their physiology. Dextransucrases are thus confronted with steadily changing reaction conditions in regards to the environmental pH, which can further affect the amount of released dextransucrases. In this work, we studied the effect of the environmental pH on the release, the productivity and the product specificity of the dextransucrase expressed by Lactobacillus (L.) hordei TMW 1.1822. Dextransucrases were recovered as crude extracts at pH 3.5-pH 6.5 and then again used to produce dextrans at these pH values. The respectively produced dextran amounts and sizes were determined and the obtained results finally systematically correlated.ResultsMaximum dextran amounts were produced at pH 4.0 and pH 4.5, while the productivity of the dextransucrases significantly decreased at pH 3.5 and pH 6.5. The distribution of dextran amounts produced at different pH most likely reflects the pH dependent activity of the dextransucrases released by L. hordei, since different transglycosylation rates were determined at different pH using the same dextransucrase amounts. Moreover, similar hydrolysis activities were detected at all tested conditions despite significant losses of transglycosylation activities indicating initial hydrolysis prior to transglycosylation reactions. The molar masses and rms radii of dextrans increased up to pH 5.5 independently of the stability of the enzyme. The gelling properties of dextrans produced at pH 4.0 and pH 5.5 were different.ConclusionsThe presented methodological approach allows the controlled production of dextrans with varying properties and could be transferred and adapted to other microbes for systematic studies on the release and functionality of native sucrases or other extracellular enzymes.

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Product release rather than chelation determines metal specificity for ferrochelatase.

Publications

Product release rather than chelation determines metal specificity for ferrochelatase.

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