Aconitase: Structure and function

Limin Zheng, Purdue University

Abstract

Aconitase is the second enzyme in the citric acid cycle. It catalyzes the reversible dehydration of citrate to cis-aconitate and the consequent hydration of cis-aconitate. A full length cDNA encoding porcine heart aconitase has been cloned and sequenced. The 2700-base pair (bp) cDNA contains a 29-bp 5$\sp\prime$ untranslated region, a 2343-bp coding segment, and a 327-bp 3$\sp\prime$ untranslated region. The porcine heart enzyme is synthesized as a precursor containing a mitochondrial targeting sequence of 27 amino acid residues which is cleaved to yield a mature enzyme of 754 amino acids, M$\sb{\rm r}$ = 82,754, having a blocked amino terminus. The NH$\sb2$-terminal pyroglutamyl residue of the mature enzyme was identified by fast atom bombardment mass spectrometry and sequence analysis of an NH$\sb2$-terminal peptide. A cDNA encoding mature porcine heart aconitase was over-expressed in Escherichia coli under the control of a phage T7 promoter. Mature 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 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 Asp$\sp{100}$, His$\sp{101}$, Asp$\sp{165}$, Arg$\sp{580}$ and Ser$\sp{642}$ result in a 10$\sp3$ to 10$\sp5$-fold drop in activity, which suggests that these residues are involved in critical aspects of the reaction. Arg$\sp{580}$ appears to be a key residue for substrate binding, as shown by a 30-fold increased K$\sb{\rm m}$ and loss of tight substrate binding. Results of mutagenesis support the interpretation of the X-ray model, namely that Asp$\sp{100}$ and His$\sp{101}$ may form an ion pair for elimination of the substrate hydroxyl and Ser$\sp{642}$ 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. Asp$\sp{165}$ appears to play a critical role in the interaction of Fe$\sb{\rm a}$ with substrate. The iron-responsive element-like RNA sequence at the 5$\sp\prime$ end of porcine mitochondrial aconitase has been demonstrated to be bound by iron-responsive element-binding protein. Bovine cytosolic aconitase has been shown to bind iron-responsive element specifically. These results suggest that cytosolic aconitase may be an iron-responsive element-binding protein, and the translation of mitochondrial aconitase mRNA may be subject to iron-dependent regulation.

Degree

Ph.D.

Advisors

Zalkin, Purdue University.

Subject Area

Biochemistry

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