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Mutational analysis of active site residues in pig heart aconitase.
J Biol Chem. 1992 Apr 15; 267(11):7895-903.JB

Abstract

A cDNA encoding mature porcine heart aconitase was over-expressed in Escherichia coli under the control of a phage T7 promoter. Recombinant aconitase purified from E. coli was identical to the enzyme from pig and beef heart in size, [3Fe-4S] and [4Fe-4S] cluster structure and enzymatic activity. Nine amino acid residues in close proximity to the Fe-S cluster and bound substrate (Lauble, H., Kennedy, M.C., Beinert, H., and Stout, C.D. (1992) Biochemistry, in press) were replaced by site-directed mutagenesis. Fe-S cluster environment as indicated by the EPR spectrum, tight binding of substrate, and enzymatic activity were compared for the mutant and wild type enzymes. Significant perturbations were detected for all of the mutant enzymes. Replacements for Asp100, His101, Asp165, Arg580, and Ser642 result in a 10(3)-10(5)-fold drop in activity, which suggests that these residues are involved in critical aspects of the reaction. Arg580 appears to be a key residue for substrate binding, as shown by a 30-fold increased Km and loss of tight substrate binding. Results of mutagenesis support the interpretation of the x-ray model, namely that Asp100 and His101 form an ion pair for elimination of the substrate hydroxyl and Ser642 may function as a general base for proton abstraction from citrate or isocitrate in the dehydration step and protonation of cis-aconitate in the hydration step. Asp165 appears to play a critical role in the interaction of Fea with substrate.

Authors+Show Affiliations

Zheng L
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153.
Kennedy MC
No affiliation info available
Beinert H
No affiliation info available
Zalkin H
No affiliation info available

MeSH

Aconitate HydrataseAmino Acid SequenceAmino AcidsAnimalsBase SequenceBinding SitesCloning, MolecularDNAElectron Spin Resonance SpectroscopyElectrophoresis, Polyacrylamide GelEscherichia coliKineticsMolecular Sequence DataMutagenesis, Site-DirectedMyocardiumPlasmidsProtein ConformationRecombinant ProteinsSwine

Pub Type(s)

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

Language

eng

PubMed ID

1313811

Citation

Zheng, L, et al. "Mutational Analysis of Active Site Residues in Pig Heart Aconitase." The Journal of Biological Chemistry, vol. 267, no. 11, 1992, pp. 7895-903.
Zheng L, Kennedy MC, Beinert H, et al. Mutational analysis of active site residues in pig heart aconitase. J Biol Chem. 1992;267(11):7895-903.
Zheng, L., Kennedy, M. C., Beinert, H., & Zalkin, H. (1992). Mutational analysis of active site residues in pig heart aconitase. The Journal of Biological Chemistry, 267(11), 7895-903.
Zheng L, et al. Mutational Analysis of Active Site Residues in Pig Heart Aconitase. J Biol Chem. 1992 Apr 15;267(11):7895-903. PubMed PMID: 1313811.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Mutational analysis of active site residues in pig heart aconitase. AU - Zheng,L, AU - Kennedy,M C, AU - Beinert,H, AU - Zalkin,H, PY - 1992/4/15/pubmed PY - 1992/4/15/medline PY - 1992/4/15/entrez SP - 7895 EP - 903 JF - The Journal of biological chemistry JO - J Biol Chem VL - 267 IS - 11 N2 - A cDNA encoding mature porcine heart aconitase was over-expressed in Escherichia coli under the control of a phage T7 promoter. Recombinant aconitase purified from E. coli was identical to the enzyme from pig and beef heart in size, [3Fe-4S] and [4Fe-4S] cluster structure and enzymatic activity. Nine amino acid residues in close proximity to the Fe-S cluster and bound substrate (Lauble, H., Kennedy, M.C., Beinert, H., and Stout, C.D. (1992) Biochemistry, in press) were replaced by site-directed mutagenesis. Fe-S cluster environment as indicated by the EPR spectrum, tight binding of substrate, and enzymatic activity were compared for the mutant and wild type enzymes. Significant perturbations were detected for all of the mutant enzymes. Replacements for Asp100, His101, Asp165, Arg580, and Ser642 result in a 10(3)-10(5)-fold drop in activity, which suggests that these residues are involved in critical aspects of the reaction. Arg580 appears to be a key residue for substrate binding, as shown by a 30-fold increased Km and loss of tight substrate binding. Results of mutagenesis support the interpretation of the x-ray model, namely that Asp100 and His101 form an ion pair for elimination of the substrate hydroxyl and Ser642 may function as a general base for proton abstraction from citrate or isocitrate in the dehydration step and protonation of cis-aconitate in the hydration step. Asp165 appears to play a critical role in the interaction of Fea with substrate. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/1313811/Mutational_analysis_of_active_site_residues_in_pig_heart_aconitase_ DB - PRIME DP - Unbound Medicine ER -