Triosephosphate isomerase (TPI) is a glycolytic enzyme that converts dihydroxyacetone phosphate (DHAP) into glyceraldehyde 3-phosphate (GAP). that enables the efficient generation of novel genetic variants. Using this system we demonstrate that can be genetically complemented by encoding a catalytically inactive enzyme. Furthermore our results demonstrate a non-metabolic function for TPI the VX-765 loss of which contributes significantly to the neurological dysfunction with this animal model. gene. TPI deficiency is clinically characterized by symptoms such as hemolytic anemia cardiomyopathy neurological dysfunction and degeneration and premature death (Schneider 2000 Orosz et al. 2006 Pathogenic TPI deficiency mutations can affect the promoter or coding sequence and all have been reported to dramatically reduced TPI activity owing to changes in catalysis and/or enzyme stability (Daar et al. 1986 Hollán et al. 1993 Arya et al. 1997 TPI deficiency has VX-765 a very poor genotype-phenotype correlation and studies to elucidate pathogenesis are extremely limited especially in animal systems. are the only model system recognized to date in which mutants have been shown to recapitulate the neurological phenotypes seen in human being individuals (Celotto et al. 2006 Gnerer et al. 2006 We have previously isolated an animal model of TPI deficiency known as is definitely characterized by shortened life-span neurodegeneration and conditional behavioral abnormalities (Celotto et al. 2006 resulting from a missense mutation causing a methionine to threonine substitution (M80T). The affected methionine is present near the dimer interface yet does not seem to result in a shift in monomer-dimer populations (Seigle et al. 2008 However the mutation offers been shown to induce irregular proteasomal degradation of TPI resulting in reduced total TPI protein (Seigle et al. 2008 Hrizo and Palladino 2010 Interestingly we have previously shown that this loss-of-function mutation can be attenuated by overexpressing mutant VX-765 TPIsgk (Celotto et al. 2006 This effect led us to query whether the presence of the enzyme or its catalytic activity was most important to the pathogenesis of disease phenotypes locus. This process establishes an founder line which can be used to modify the gene locus using highly efficient transgenesis. We hypothesized that if the presence of the enzyme was essential to pathogenesis self-employed of catalytic activity we would be able to rescue the disease phenotypes having a catalytically inactive variant of the protein. Lys11 of TPI is definitely a fully conserved catalytic residue known to be required for substrate binding and substitution to Met completely abolishes catalysis (Lodi et al. 1994 Wierenga et al. 2010 We have generated the founder line and have used GE to produce genetically matches the longevity and behavior of the animal model of TPI deficiency. Furthermore catalytically inactive TPI matches phenotypes without IL5R enhancing its stability catalysis or reducing the connected metabolic stress. Collectively these data suggest a function of TPI self-employed of its catalytic activity which is vital to behavior and longevity. Results Recombinant TPI enzyme activity Earlier studies established that is a recessive loss-of-function mutation characterized by reduced TPI levels (Seigle et al. 2008 Genetic data suggested that TPIsgk retained adequate function to save mutant survival and behavioral phenotypes if overexpressed (Seigle et al. 2008 These data led us to hypothesize that reduced TPI catalysis was essential to the pathogenesis of TPI deficiency. To investigate this hypothesis further we generated recombinant WT (dWT) and TPIsgk (dM80T) and examined the kinetics of isomerase activity for each enzyme (Table?1). These data demonstrate that TPIsgk (dM80T) exhibits a substantial reduction in isomerase activity. The dM80T protein has a 33% decrease in substrate affinity and ～11-fold reduction in catalytic activity compared to WT enzyme. This ultimately resulted in a ～15-collapse reduction in enzyme effectiveness. Both enzymes displayed standard Michaelis-Menten kinetics (supplementary material VX-765 Fig. S1). Table 1. Kinetic guidelines of wild-type (dWT) and (dM80T) triosephosphate isomerase enzymes To assess the role of the M80 position within TPI function we analyzed the crystal structure of TPI from (Zhang et al. 1994 – TPI.