Altered orientation of active site residues in variants of human ferrochelatase. Evidence for a hydrogen bond network involved in catalysis

Harry A. Dailey, Chia Kuei Wu, Peter Horanyi, Amy Elizabeth Medlock, Wided Najahi-Missaoui, Amy E. Burden, Tamara A. Dailey, John Rose

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Ferrochelatase catalyzes the terminal step in heme biosynthesis, the insertion of ferrous iron into protoporphyrin to form protoheme IX. The crystal structures of human ferrochelatase both with and without the protoporphyrin substrate bound have been determined previously. The substrate-free enzyme has an open active site pocket, while in the substrate-bound enzyme, the active site pocket is closed around the porphyrin macrocycle and a number of active site residues have reoriented side chains. To understand how and why these structural changes occur, we have substituted three amino acid residues (H263, H341, and F337) whose side chains occupy different spatial positions in the substrate-free versus substrate-bound ferrochelatases. The catalytic and structural properties of ferrochelatases containing the amino acid substitutions H263C, H341C, and F337A were examined. It was found that in the H263C and H341C variants, but not the F337A variant enzymes, the side chains of N75, M76, R164, H263, F337, H341, and E343 are oriented in a fashion similar to what is found in ferrochelatase with the bound porphyrin substrate. However, all of the variant forms possess open active site pockets which are found in the structure of porphyrin-free ferrochelatase. Thus, while the interior walls of the active site pocket are remodeled in these variants, the exterior lips remain unaltered in position. One possible explanation for this collective reorganization of active site side chains is the presence of a hydrogen bond network among H263, H341, and E343. This network is disrupted in the variants by alteration of H263C or H341C. In the substrate-bound enzyme, the formation of a hydrogen bond between H263 and a pyrrole nitrogen results in disruption of the network. The possible role of this network in catalysis is discussed.

Original languageEnglish (US)
Pages (from-to)7973-7979
Number of pages7
JournalBiochemistry
Volume46
Issue number27
DOIs
StatePublished - Jul 10 2007
Externally publishedYes

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Ferrochelatase
Catalysis
Hydrogen
Catalytic Domain
Hydrogen bonds
Porphyrins
Substrates
Enzymes
Heme
Pyrroles
Amino Acids
Amino Acid Substitution
Lip
Biosynthesis
Nitrogen
Iron
Structural properties
Substitution reactions
Crystal structure

ASJC Scopus subject areas

  • Biochemistry

Cite this

Dailey, H. A., Wu, C. K., Horanyi, P., Medlock, A. E., Najahi-Missaoui, W., Burden, A. E., ... Rose, J. (2007). Altered orientation of active site residues in variants of human ferrochelatase. Evidence for a hydrogen bond network involved in catalysis. Biochemistry, 46(27), 7973-7979. https://doi.org/10.1021/bi700151f

Altered orientation of active site residues in variants of human ferrochelatase. Evidence for a hydrogen bond network involved in catalysis. / Dailey, Harry A.; Wu, Chia Kuei; Horanyi, Peter; Medlock, Amy Elizabeth; Najahi-Missaoui, Wided; Burden, Amy E.; Dailey, Tamara A.; Rose, John.

In: Biochemistry, Vol. 46, No. 27, 10.07.2007, p. 7973-7979.

Research output: Contribution to journalArticle

Dailey, HA, Wu, CK, Horanyi, P, Medlock, AE, Najahi-Missaoui, W, Burden, AE, Dailey, TA & Rose, J 2007, 'Altered orientation of active site residues in variants of human ferrochelatase. Evidence for a hydrogen bond network involved in catalysis', Biochemistry, vol. 46, no. 27, pp. 7973-7979. https://doi.org/10.1021/bi700151f
Dailey, Harry A. ; Wu, Chia Kuei ; Horanyi, Peter ; Medlock, Amy Elizabeth ; Najahi-Missaoui, Wided ; Burden, Amy E. ; Dailey, Tamara A. ; Rose, John. / Altered orientation of active site residues in variants of human ferrochelatase. Evidence for a hydrogen bond network involved in catalysis. In: Biochemistry. 2007 ; Vol. 46, No. 27. pp. 7973-7979.
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abstract = "Ferrochelatase catalyzes the terminal step in heme biosynthesis, the insertion of ferrous iron into protoporphyrin to form protoheme IX. The crystal structures of human ferrochelatase both with and without the protoporphyrin substrate bound have been determined previously. The substrate-free enzyme has an open active site pocket, while in the substrate-bound enzyme, the active site pocket is closed around the porphyrin macrocycle and a number of active site residues have reoriented side chains. To understand how and why these structural changes occur, we have substituted three amino acid residues (H263, H341, and F337) whose side chains occupy different spatial positions in the substrate-free versus substrate-bound ferrochelatases. The catalytic and structural properties of ferrochelatases containing the amino acid substitutions H263C, H341C, and F337A were examined. It was found that in the H263C and H341C variants, but not the F337A variant enzymes, the side chains of N75, M76, R164, H263, F337, H341, and E343 are oriented in a fashion similar to what is found in ferrochelatase with the bound porphyrin substrate. However, all of the variant forms possess open active site pockets which are found in the structure of porphyrin-free ferrochelatase. Thus, while the interior walls of the active site pocket are remodeled in these variants, the exterior lips remain unaltered in position. One possible explanation for this collective reorganization of active site side chains is the presence of a hydrogen bond network among H263, H341, and E343. This network is disrupted in the variants by alteration of H263C or H341C. In the substrate-bound enzyme, the formation of a hydrogen bond between H263 and a pyrrole nitrogen results in disruption of the network. The possible role of this network in catalysis is discussed.",
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AU - Medlock, Amy Elizabeth

AU - Najahi-Missaoui, Wided

AU - Burden, Amy E.

AU - Dailey, Tamara A.

AU - Rose, John

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N2 - Ferrochelatase catalyzes the terminal step in heme biosynthesis, the insertion of ferrous iron into protoporphyrin to form protoheme IX. The crystal structures of human ferrochelatase both with and without the protoporphyrin substrate bound have been determined previously. The substrate-free enzyme has an open active site pocket, while in the substrate-bound enzyme, the active site pocket is closed around the porphyrin macrocycle and a number of active site residues have reoriented side chains. To understand how and why these structural changes occur, we have substituted three amino acid residues (H263, H341, and F337) whose side chains occupy different spatial positions in the substrate-free versus substrate-bound ferrochelatases. The catalytic and structural properties of ferrochelatases containing the amino acid substitutions H263C, H341C, and F337A were examined. It was found that in the H263C and H341C variants, but not the F337A variant enzymes, the side chains of N75, M76, R164, H263, F337, H341, and E343 are oriented in a fashion similar to what is found in ferrochelatase with the bound porphyrin substrate. However, all of the variant forms possess open active site pockets which are found in the structure of porphyrin-free ferrochelatase. Thus, while the interior walls of the active site pocket are remodeled in these variants, the exterior lips remain unaltered in position. One possible explanation for this collective reorganization of active site side chains is the presence of a hydrogen bond network among H263, H341, and E343. This network is disrupted in the variants by alteration of H263C or H341C. In the substrate-bound enzyme, the formation of a hydrogen bond between H263 and a pyrrole nitrogen results in disruption of the network. The possible role of this network in catalysis is discussed.

AB - Ferrochelatase catalyzes the terminal step in heme biosynthesis, the insertion of ferrous iron into protoporphyrin to form protoheme IX. The crystal structures of human ferrochelatase both with and without the protoporphyrin substrate bound have been determined previously. The substrate-free enzyme has an open active site pocket, while in the substrate-bound enzyme, the active site pocket is closed around the porphyrin macrocycle and a number of active site residues have reoriented side chains. To understand how and why these structural changes occur, we have substituted three amino acid residues (H263, H341, and F337) whose side chains occupy different spatial positions in the substrate-free versus substrate-bound ferrochelatases. The catalytic and structural properties of ferrochelatases containing the amino acid substitutions H263C, H341C, and F337A were examined. It was found that in the H263C and H341C variants, but not the F337A variant enzymes, the side chains of N75, M76, R164, H263, F337, H341, and E343 are oriented in a fashion similar to what is found in ferrochelatase with the bound porphyrin substrate. However, all of the variant forms possess open active site pockets which are found in the structure of porphyrin-free ferrochelatase. Thus, while the interior walls of the active site pocket are remodeled in these variants, the exterior lips remain unaltered in position. One possible explanation for this collective reorganization of active site side chains is the presence of a hydrogen bond network among H263, H341, and E343. This network is disrupted in the variants by alteration of H263C or H341C. In the substrate-bound enzyme, the formation of a hydrogen bond between H263 and a pyrrole nitrogen results in disruption of the network. The possible role of this network in catalysis is discussed.

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