Human triose-phosphate isomerase deficiency: a single amino acid substitution results in a thermolabile enzyme.

Abstract

Triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1) deficiency is a recessive disorder that results in hemolytic anemia and neuromuscular dysfunction. To determine the molecular basis of this disorder, a TPI allele from two unrelated patients homozygous for TPI deficiency was compared with an allele from a normal individual. Each disease-associated sequence harbors a G-C -+ C-G transversion in the codon for amino acid-104 and specifies a structurally altered protein in which a glutamate residue is replaced by an aspartate residue. The importance of glutamate-104 to enzyme structure and function is implicated by its conservation in the TPI protein of all species that have been characterized to date. The glutamate-toaspartate substitution results in a thermolabile enzyme as demonstrated by assaysof-TPI activity in cultured fibroblasts of each patient and cultured Chinese hamster ovary (CHO) cells that were stably transformed with the mutant alleles. Although this substitution conserves the overall charge of amino acid-104, the x-ray crystal structure of chicken TPI indicates that the loss of a side-chain methylene group (-CH2CH2COO -* -CH2COO ) is sufficient to disrupt the counterbalancing of charges that normally exists within a hydrophobic pocket of the native enzyme.Triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1) catalyzes the movement of a single proton to interconvert dihydroxyacetone phosphate and glyceraldehyde 3-phosphate in glycolysis and gluconeogenesis (1). The enzyme, a dimer of identical subunits that are 248 amino acids in humans (2, 3), has no cofactors, required metal ions, or cooperativity between subunits. Both prokaryotic and eukaryotic TPI are very similar in sequence and structure and are characterized by a high catalytic efficiency. The second-order rate constant for the formation of dihydroxyacetone phosphate, the thermodynamically favored reaction, is close to the rate constant for a diffusion-limited encounter of substrate and enzyme (4, 5).Site-directed mutagenesis combined with crystallographic and kinetic studies have provided a means of defining the contribution of specific TPI amino acids to enzyme activity (6, 7). This method has focused on amino acids implicated by high-resolution structural data and chemical-modification experiments to be important to the catalytic mechanism of the enzyme. We have chosen a complementary approach to understand TPI structure-function relationships in studying natural TPI gene mutations that exist within the human population. Hereditary TPI deficiency is an autosomal recessive disease that has severe clinical manifestations including chronic hemolytic anemia and neuromuscular disorders (8). Homozygous-deficient individuals usually have 3-20% of normal TPI activity; all of this activity is heat labile, suggesting that at least one allele encodes a structurally altered protein (3, 9, 10). The other allele is presumably null, since mutations producing no detectable ...

Drosophila Model of Human Inherited Triosephosphate Isomerase Deficiency Glycolytic Enzymopathy

“…Human TPI disease conditions are typically caused by specific homozygous missense mutations that often encode thermolabile proteins (Daar et al 1986;Chang et al 1993). The recessive nature and temperature sensitivity of the sgk 1 mutant, whose phenotypes are closely analogous to human disease symptoms, suggest that this mutant will effectively model TPI enzymopathic disease and provide an amenable genetic model system for studying the basic mechanisms of disease pathogenesis.…”

“…At least nine different TPI missense mutations that affect various positions throughout the encoded protein have been identified as the cause of glycolytic enzymopathies (Daar et al 1986;Daar and Maquat 1988;Chang et al 1993;Watanabe et al 1996;Arya et al 1997;Valentin et al 2000) (supplemental Figure S1 at http://www.genetics.org/supplemental/). Heterozygosity of null human TPI alleles has been observed in 5% of African American and 0.5% of Caucasian populations, although the disease is typically caused by homozygous missense mutations (Mohrenweiser and Fielek 1982;Mohrenweiser 1987).…”

“…Our studies demonstrate that reduced longevity and progressive neural degeneration are associated with the temperature-sensitive sgk 1 fly mutation. Interestingly, some of the well-characterized mutations that cause human TPI deficiency disease have been shown to be temperature sensitive, owing to thermal instability of the encoded protein (Daar et al 1986;Chang et al 1993). Although TPI has a well-characterized biochemical function, the mechanism of disease pathogenesis is unclear.…”

Triosephosphate isomerase deficiency: Facts and doubts

“…The amino acid substitution results in loss of activity due to the instability of the mutant enzyme, which likely results from the dissociation of the active dimers into inactive monomers (24). The instability of this mutant enzyme was demonstrated by assays of TPI activity in cultured fibroblasts from patients and in cultured Chinese hamster ovary (CHO) cells that were stably transformed with mutant alleles (5).…”

Degradation of Functional Triose Phosphate Isomerase Protein UnderliessugarkillPathology

“…This suggests that TPI sugarkill animals may serve as a useful model of TPI impairment where varying temperatures can be utilized to study a series of hypomorphic conditions from mild to severe. Furthermore, in vitro temperature sensitivity has also been reported for the most common homozygous human mutant allele, Glu104Asp (Daar et al 1986;Arya et al 1997), suggesting a shared mechanism of pathogenesis exists with at least one of the common disease-causing missense mutations.…”