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. 2015 Jan;1852(1):61-9.
doi: 10.1016/j.bbadis.2014.10.010. Epub 2014 Oct 16.

Triosephosphate Isomerase I170V Alters Catalytic Site, Enhances Stability and Induces Pathology in a Drosophila Model of TPI Deficiency

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Triosephosphate Isomerase I170V Alters Catalytic Site, Enhances Stability and Induces Pathology in a Drosophila Model of TPI Deficiency

Bartholomew P Roland et al. Biochim Biophys Acta. .
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Abstract

Triosephosphate isomerase (TPI) is a glycolytic enzyme which homodimerizes for full catalytic activity. Mutations of the TPI gene elicit a disease known as TPI Deficiency, a glycolytic enzymopathy noted for its unique severity of neurological symptoms. Evidence suggests that TPI Deficiency pathogenesis may be due to conformational changes of the protein, likely affecting dimerization and protein stability. In this report, we genetically and physically characterize a human disease-associated TPI mutation caused by an I170V substitution. Human TPI(I170V) elicits behavioral abnormalities in Drosophila. An examination of hTPI(I170V) enzyme kinetics revealed this substitution reduced catalytic turnover, while assessments of thermal stability demonstrated an increase in enzyme stability. The crystal structure of the homodimeric I170V mutant reveals changes in the geometry of critical residues within the catalytic pocket. Collectively these data reveal new observations of the structural and kinetic determinants of TPI Deficiency pathology, providing new insights into disease pathogenesis.

Keywords: Drosophila; Structure; Triosephosphate isomerase; Triosephosphate isomerase deficiency.

Figures

Fig. 1
Fig. 1
hTPII170V is characterized by behavioral dysfunction but not reduced longevity. hTPII170V/TPInull exhibits mechanical (A) and thermal (B) stress sensitivity relative to hTPIWT/TPInull, n≥20. Conversely, hTPII170V/TPInull demonstrated similar lifespans (C) as hTPIWT/TPInull, n≥89. * indicates p<0.05, ** p<0.01, and *** p<0.001. Error bars indicate S.E.M.
Fig. 2
Fig. 2
hTPII170V exhibits reduced catalysis and normal cellular TPI levels. Isomerase assays reveal hTPII170V/TPInull reduces lysate TPI activity relative to hTPIWT/TPInull (A), n=3. Independent samples of hTPIWT/TPInull and hTPII170V/TPInull (#1,#2,#3) demonstrate similar levels of cellular TPI (C), with quantification (B), n=3. ns indicates no significance. Error bars represent S.E.M.
Fig. 3
Fig. 3
hTPII170V reduces Km and catalytic turnover. hTPIWT and hTPII170V display typical Michaelis Menten kinetic profiles (A,B). I170V reduces Km and catalytic turnover (C). Error bars and ± indicate S.E.M.
Fig. 4
Fig. 4
I170V enhances TPI stability relative to WT. CD thermal shift analyses demonstrate a stabilization of TPI due to the I170V substitution (A), and responsiveness to catalytic site occupancy via DL-GP substrate analog (A). Far-UV spectra demonstrate similar folding between hTPIWT (B) and hTPII170V (C), with averaged spectra from selected temperatures (B,C). No refolding was noted in either enzyme. Error bars represent S.D.
Fig. 5
Fig. 5
Architecture of WT and hTPII170V. (A) Overview of wild-type hTPI dimer with one subunit indicated as a solid grey surface while the other is represented in both cartoon (blue) and semitransparent surface (white). The position of the loop 6, which forms the lid and completes the active site pocket is indicated in magenta. The canonical nomenclature for landmark secondary structure elements is indicated. (B) The positions of bromide and phosphate ions observed in hTPIWT is indicated. The position of the substrate analog PGA within the catalytic pocket as positioned via structural alignment of TPI is indicated (PDB: 1HTI) [48]. Only the PGA from this alignment is shown for clarity. The network of hydrogen bonding connecting critical catalytic residues are indicated by green dashes.
Fig. 6
Fig. 6
The I170V substitution decouples S96 positioning from lid closure. (A) Substitution of valine at position 170 does not alter E165 or loop 6 positioning within the closed lid state. The structures of hTPII170V (yellow), PGH-liganded TPI (PDB 1TRD, grey), and TPI in an unliganded open state (PDB 2JK2, green) were superposed and the positions of E165 and I170 are indicated (B). Repositioning of S96 within a closed lid structure in I170V. View of the catalytic pocket after structural alignment of WT (Blue) and I170V (Gold) hTPI structures. The catalytically important residues S96, H95, and N11 are shown as sticks. The catalytic residue E165 is unchanged and is omitted for clarity. The lid for both structures are indicated in magenta. (C) hTPII170V adopts a closed lid conformation. The active site from a structural alignment of hTPIWT (green) in an open lid conformation (2JK2) [4] and hTPII170V (gold) is shown. The positions of the lids and respective active site residues are indicated.

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