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. 2002 Jun;11(6):1552-7.
doi: 10.1110/ps.0201502.

X-ray crystallographic and kinetic correlation of a clinically observed human fumarase mutation

Marcel Estévez  1 , Jeremy Skarda, Josh Spencer, Leonard Banaszak, Todd M Weaver
Affiliations

X-ray crystallographic and kinetic correlation of a clinically observed human fumarase mutation

Marcel Estévez et al. Protein Sci. 2002 Jun.

Abstract

Fumarase catalyzes the reversible conversion of fumarate to S- malate during the operation of the ubiquitous Kreb's cycle. Previous studies have shown that the active site includes side chains from three of the four subunits within the tetrameric enzyme. We used a clinically observed human mutation to narrow our search for potential catalytic groups within the fumarase active site. Offspring homozygous for the missense mutation, a G-955-C transversion in the fumarase gene, results in the substitution of a glutamine at amino acid 319 for the normal glutamic acid. To more fully understand the implications of this mutation, a single-step site-directed mutagenesis method was used to generate the homologous substitution at position 315 within fumarase C from Escherichia coli. Subsequent kinetic and X-ray crystal structure analyses show changes in the turnover number and the cocrystal structure with bound citrate.

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Figures

Fig. 1.
Fig. 1.
Superfamily 300s loop sequence alignment. The sequence alignment shows identity within the 300s loop region of the superfamily members, fumarase, aspartase, adenylosuccinate lyase, arginosuccinate lyase, and δ-crystallin. Each distinct family of sequences has been grouped and identified within the alignment. The alignment is numbered based on E. coli fumarase C, where the italicized region identifies the signature sequence spanning Gly 317 through Glu 331. (↓) Glu 315; (*) identical amino acids within the superfamily signature sequence; (Yeast) Saccharomyces cerevisiae; (E. coli) Escherichia coli; (S. marcescens) Serratia marcescens; (P. fluorescens) Pseudomonas fluorescens; (B. subtilis) Bacillus subtilis.
Fig. 2.
Fig. 2.
Structural comparisons between native and E315Q fumarase active sites. (A) The native FumC active site illustrating the positions of Glu 315, citrate, and the highly coordinated active-site water (W54). (B) The E315Q active site with the positions of Gln 315 and citrate and the absence of W54. Dashed lines are hydrogen bonds. Atoms are shaded as follows: carbon, light gray; oxygen, black; and nitrogen, dark gray. Amino acids are labeled using the one-letter code with E, glutamic acid; K, lysine; N, asparagines; Q, glutamine; S, serine; T, threonine; and H, histidine. Subunit designation is provided by the lowercase letter following the amino acid number.
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