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Steric and conformational features of the aconitase mechanism.
Proteins. 1995 May; 22(1):1-11.P

Abstract

Crystal structures of mitochondrial aconitase with alpha-methylisocitrate and with sulfate bound have been solved and refined at 2.0 A resolution with R factors of 18.2 and 16.8%, respectively. The steric factors and conformational effects observed in both new structures support the proposed mechanism for the overall reaction catalyzed by aconitase. The alternate substrate alpha-methylisocitrate is derived from alpha-methyl-cis-aconitate during crystallization and is observed to bind in the active site in a manner very similar to that observed for isocitrate. The methyl group is accommodated by favorable contact with Ile-425. However, the other potential hydration product of alpha-methyl-cis-aconitate, alpha-methylcitrate, cannot be accommodated in the active site due to steric conflict of the methyl group with Asp-165. The results are consistent with the requirement that cis-aconitate must bind in two ways, in the citrate mode and in the isocitrate mode. Crystals of aconitase with sulfate bound are isomorphous to those with isocitrate bound. However, the structure displays significant conformational changes, providing a model for the substrate-free state of enzyme. Three water molecules bind in place of the C alpha- and C beta-hydroxyl and carboxyl groups of isocitrate, while sulfate binds in place of the C gamma-carboxyl group. Side chains of Ser-642 and Arg-447 in the active site rotate to pair with other side chains in the absence of substrate. The new conformation of Arg-447 triggers a concerted set of shifts which transmits conformational change to the surface of the protein, 30 A from the active site. In the absence of substrate, a chain segment containing the [4Fe-4S] ligand Cys-358 also shifts, resulting in the net translation and reorientation of the Fe-S cluster.

Authors+Show Affiliations

Institut für Organische Chemie, Universität Stuttgart, Germany.No affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

7675781

Citation

Lauble, H, and C D. Stout. "Steric and Conformational Features of the Aconitase Mechanism." Proteins, vol. 22, no. 1, 1995, pp. 1-11.
Lauble H, Stout CD. Steric and conformational features of the aconitase mechanism. Proteins. 1995;22(1):1-11.
Lauble, H., & Stout, C. D. (1995). Steric and conformational features of the aconitase mechanism. Proteins, 22(1), 1-11.
Lauble H, Stout CD. Steric and Conformational Features of the Aconitase Mechanism. Proteins. 1995;22(1):1-11. PubMed PMID: 7675781.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Steric and conformational features of the aconitase mechanism. AU - Lauble,H, AU - Stout,C D, PY - 1995/5/1/pubmed PY - 1995/5/1/medline PY - 1995/5/1/entrez SP - 1 EP - 11 JF - Proteins JO - Proteins VL - 22 IS - 1 N2 - Crystal structures of mitochondrial aconitase with alpha-methylisocitrate and with sulfate bound have been solved and refined at 2.0 A resolution with R factors of 18.2 and 16.8%, respectively. The steric factors and conformational effects observed in both new structures support the proposed mechanism for the overall reaction catalyzed by aconitase. The alternate substrate alpha-methylisocitrate is derived from alpha-methyl-cis-aconitate during crystallization and is observed to bind in the active site in a manner very similar to that observed for isocitrate. The methyl group is accommodated by favorable contact with Ile-425. However, the other potential hydration product of alpha-methyl-cis-aconitate, alpha-methylcitrate, cannot be accommodated in the active site due to steric conflict of the methyl group with Asp-165. The results are consistent with the requirement that cis-aconitate must bind in two ways, in the citrate mode and in the isocitrate mode. Crystals of aconitase with sulfate bound are isomorphous to those with isocitrate bound. However, the structure displays significant conformational changes, providing a model for the substrate-free state of enzyme. Three water molecules bind in place of the C alpha- and C beta-hydroxyl and carboxyl groups of isocitrate, while sulfate binds in place of the C gamma-carboxyl group. Side chains of Ser-642 and Arg-447 in the active site rotate to pair with other side chains in the absence of substrate. The new conformation of Arg-447 triggers a concerted set of shifts which transmits conformational change to the surface of the protein, 30 A from the active site. In the absence of substrate, a chain segment containing the [4Fe-4S] ligand Cys-358 also shifts, resulting in the net translation and reorientation of the Fe-S cluster. SN - 0887-3585 UR - https://www.unboundmedicine.com/medline/citation/7675781/Steric_and_conformational_features_of_the_aconitase_mechanism_ L2 - https://doi.org/10.1002/prot.340220102 DB - PRIME DP - Unbound Medicine ER -