Probing the role of the fully conserved Cys126 in triosephosphate isomerase by site-specific mutagenesis--distal effects on dimer stability

Cys126 is a completely conserved residue in triosephosphate isomerase that is proximal to the active site but has been ascribed no specific role in catalysis. A previous study of the C126S and C126A mutants of yeast TIM reported substantial catalytic activity for the mutant enzymes, leading to the suggestion that this residue is implicated in folding and stability [Gonzalez-Mondragon E et al. (2004) Biochemistry 43, 3255-3263]. We re-examined the role of Cys126 with the Plasmodium falciparum enzyme as a model. Five mutants, C126S, C126A, C126V, C126M, and C126T, were characterized. Crystal structures of the 3-phosphoglycolate-bound C126S mutant and the unliganded forms of the C126S and C126A mutants were determined at a resolution of 1.7-2.1 ?. Kinetic studies revealed an approximately five-fold drop in k(cat) for the C126S and C126A mutants, whereas an approximately 10-fold drop was observed for the other three mutants. At ambient temperature, the wild-type enzyme and all five mutants showed no concentration dependence of activity. At higher temperatures (> 40 °C), the mutants showed a significant concentration dependence, with a dramatic loss in activity below 15 μM. The mutants also had diminished thermal stability at low concentration, as monitored by far-UV CD. These results suggest that Cys126 contributes to the stability of the dimer interface through a network of interactions involving His95, Glu97, and Arg98, which form direct contacts across the dimer interface.     

Segmental movement: definition of the structural requirements for loop closure in catalysis by triosephosphate isomerase.

To determine what drives the closure of the active-site loop in the reaction catalyzed by triosephosphate isomerase, several residues involved in hydrogen bonding between the loop and the bulk of the protein have been altered. It was known from earlier work that the loop serves two functions: to stabilize the reaction intermediate (and the two transition states that flank it) and to prevent the loss of this unstable species into free solution. To discover what elements of the protein are necessary for proper closure of the loop, selective destabilization of the "open" and the "closed" forms of the enzyme with respect to one another has been attempted. The mutant Y164F isomerase has been prepared to evaluate the importance of the structure of the "open" form, and the mutant E129Q, Y208F, and S211A enzymes have allowed investigation of the "closed" form. The integrity of the loop itself has been destabilized by making the T172A isomerase. We have found that only those mutations that destabilize the "closed" form of the enzyme significantly perturb the catalytic properties of the isomerase. The second-order rate constants (kcat/Km) of the S211A and E129Q enzymes are reduced 30-fold, and that of the mutant Y208F enzyme is reduced 2000-fold, from the level of the wild-type enzyme. The dramatic drop in activity of the Y208F enzyme is accompanied by a 200-fold increase in the dissociation constant of the intermediate analogue phosphoglycolohydroxamate. The most important property of the mobile loop of triosephosphate isomerase lies, therefore, in the stability of the system when the active site contains ligand and the loop is closed.     

Active site properties of monomeric triosephosphate isomerase (monoTIM) as deduced from mutational and structural studies.

MonoTIM is a stable monomeric variant of the dimeric trypanosomal enzyme triose phosphate isomerase (TIM) with less, but significant, catalytic activity. It is known that in TIM, three residues, Lys 13 (loop 1), His 95 (loop 4), and Glu 167 (loop 6) are the crucial catalytic residues. In the wild-type TIM dimer, loop 1 and loop 4 are very rigid because of tight interactions with residues of the other subunit. Previous structural studies indicate that Lys 13 and His 95 have much increased conformational flexibility in monoTIM. Using site-directed mutagenesis, it is shown here that Lys 13 and His 95 are nevertheless essential for optimal catalysis by monoTIM: monoTIM-K13A is completely inactive, although it can still bind substrate analogues, and monoTIM-H95A is 50 times less active. The best inhibitors of wild-type TIM are phosphoglycolohydroxamate (PGH) and 2-phosphoglycolate (2PG), with KI values of 8 microM and 26 microM, respectively. The affinity of the monoTIM active site for PGH has been reduced approximately 60-fold, whereas for 2PG, only a twofold weakening of affinity is observed. The mode of binding, as determined by protein crystallographic analysis of these substrate analogues, shows that, in particular, 2PG interacts with Lys 13 and His 95 in a way similar but not identical to that observed for the wild-type enzyme. This crystallographic analysis also shows that Glu 167 has the same interactions with the substrate analogues as in the wild type. The data presented suggest that, despite the absence of the second subunit, monoTIM catalyzes the interconversion of D-glyceraldehyde-3-phosphate and dihydroxyacetone phosphate via the same mechanism as in the wild type.     

Subunit interface of triosephosphate isomerase: site-directed mutagenesis and characterization of the altered enzyme

We have replaced asparagine residues at the subunit interface of yeast triosephosphate isomerase (TIM) using site-directed mutagenesis in order to elucidate the effects of substitutions on the catalytic activity and conformational stability of the enzyme. The mutant proteins were expressed in a strain of Escherichia coli lacking the bacterial isomerase and purified by ion-exchange and immunoadsorption chromatography. Single replacements of Asn-78 by either Thr or Ile residues had little effect on the enzyme's catalytic efficiency, while the single replacement Asn-78----Asp-78 and the double replacement Asn-14/Asn-78----Thr-14/Ile-78 appreciably lowered kcat for the substrate D-glyceraldehyde 3-phosphate. The isoelectric point of the mutant Asn-78----Asp-78 was equivalent to that of wild-type yeast TIM that had undergone a single, heat-induced deamidation, and this mutant enzyme was less resistant than wild-type TIM to denaturation and inactivation caused by elevated temperature, denaturants, tetrabutylammonium bromide, alkaline pH, and proteases.     

Developing a new drug against trichomoniasis,new inhibitory compounds of the protein triosephosphate isomerase

Trichomonas vaginalis is the protozoan parasite responsible for the most prevalent, non-viral, sexually transmitted disease, which affects millions of people around the world. The main treatment against this disease is metronidazole and some other nitroimidazole derivatives. However, between five and 20% of clinical cases of trichomoniasis are caused by parasites resistant to these drugs. Here we present three compounds that were selected using an innovative strategy, to propose them as possible drugs to combat trichomoniasis, using the glycolytic enzyme triose phosphate isomerase (TvTIM) as the drug target. In the genome of Trichomonas vaginalis there are two genes that encode for two isoforms of TvTIM, known as TvTIM1 and TvTIM2, varying by four out of 254 aminoacid residues. In this study, we used high-throughput virtual screening to search molecules that bind specifically to TvTIM isoforms, in which 34 compounds were selected from a library of nearly 450,000 compounds. The effects of the 34 compounds on the conformation and enzymatic activity of both TvTIM isoforms and their human homolog (HsTIM) were evaluated. We found three compounds that bind specifically, modify the conformation and inhibit TvTIM2 only; although the sequence of both isoforms of TvTIM is almost identical. The selectivity of these compounds towards TvTIM2 is explained by the lower conformational stability of this isoform and that these interactions can inhibit the activity of this enzyme and have an effect against this parasite. These compounds represent promising alternatives for the development of new therapeutic strategies against trichomoniasis.     

Closed conformation of the active site loop of rabbit muscle triosephosphate isomerase in the absence of substrate: evidence of conformational heterogeneity

The active site loop of triosephosphate isomerase (TIM) exhibits a hinged-lid motion, alternating between the two well defined "open" and "closed" conformations. Until now the closed conformation had only been observed in protein complexes with substrate analogues. Here, we present the first rabbit muscle apo TIM structure, refined to 1.5A resolution, in which the active site loop is either in the open or in the closed conformation in different subunits of the enzyme. In the closed conformation described here, the lid loop residues participate in stabilizing hydrogen bonds characteristic of holo TIM structures, whereas chemical interactions observed in the open loop conformation are similar to those found in the apo structures of TIM. In the closed conformation, a number of water molecules are observed at the projected ligand atom positions that are hydrogen bonded to the active site residues. Additives used during crystallization (DMSO and Tris molecules and magnesium atoms) were modeled in the electron density maps. However, no specific binding of these molecules is observed at, or close to, the active site and the lid loop. To further investigate this unusual closed conformation of the apo enzyme, two more rabbit muscle TIM structures, one in the same and another in a different crystal form, were determined. These structures present the open lid conformation only, indicating that the closed conformation cannot be explained by crystal contact effects. To rationalize why the active site loop is closed in the absence of ligand in one of the subunits, extensive comparison with previously solved TIM structures was carried out, supported by the bulk of available experimental information about enzyme kinetics and reaction mechanism of TIM. The observation of both open and closed lid conformations in TIM crystals might be related to a persistent conformational heterogeneity of this protein in solution.     

In silico studies on the substrate specificity of an l-arabinose isomerase from Bacillus licheniformis

l-Arabinose isomerase (BLAI) from Bacillus licheniformis was found to be active only with l-arabinose, unlike other l-arabinose isomerases (l-AIs) active with a variety of aldoses. Therefore, the differences in molecular interactions and substrate orientation in the active site of l-AIs have been examined and the residue at position 346 is proposed to be responsible for the unique substrate specificity of BLAI.     

Structural and mutagenesis studies of leishmania triosephosphate isomerase: a point mutation can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power

The dimeric enzyme triosephosphate isomerase (TIM) has a very tight and rigid dimer interface. At this interface a critical hydrogen bond is formed between the main chain oxygen atom of the catalytic residue Lys13 and the completely buried side chain of Gln65 (of the same subunit). The sequence of Leishmania mexicana TIM, closely related to Trypanosoma brucei TIM (68% sequence identity), shows that this highly conserved glutamine has been replaced by a glutamate. Therefore, the 1.8 A crystal structure of leishmania TIM (at pH 5.9) was determined. The comparison with the structure of trypanosomal TIM shows no rearrangements in the vicinity of Glu65, suggesting that its side chain is protonated and is hydrogen bonded to the main chain oxygen of Lys13. Ionization of this glutamic acid side chain causes a pH-dependent decrease in the thermal stability of leishmania TIM. The presence of this glutamate, also in its protonated state, disrupts to some extent the conserved hydrogen bond network, as seen in all other TIMs. Restoration of the hydrogen bonding network by its mutation to glutamine in the E65Q variant of leishmania TIM results in much higher stability; for example, at pH 7, the apparent melting temperature increases by 26 degrees C (57 degrees C for leishmania TIM to 83 degrees C for the E65Q variant). This mutation does not affect the kinetic properties, showing that even point mutations can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power at the mesophilic temperature.     

Recombinant tyrosine aminotransferase from Trypanosoma cruzi: structural characterization and site directed mutagenesis of a broad substrate specificity enzyme

The gene encoding tyrosine aminotransferase (TAT, EC 2.6.1.5) from the parasitic protozoan Trypanosoma cruzi was amplified from genomic DNA, cloned into the pET24a expression vector and functionally expressed as a C-terminally His-tagged protein in Escherichia coli BL21(DE3)pLysS. Purified recombinant TAT exhibited identical electrophoretic and enzymatic properties as the authentic enzyme from T. cruzi. Both recombinant and authentic T. cruzi TATs were highly resistant to limited tryptic cleavage and contained no disulfide bonds. Comprehensive analysis of its substrate specificity demonstrated TAT to be a broad substrate aminotransferase, with leucine, methionine as well as tyrosine, phenylalanine, tryptophan and alanine being utilized efficiently as amino donors. Valine, isoleucine and dicarboxylic amino acids served as poor substrates while polar aliphatic amino acids could not be transaminated. TAT also accepted several 2-oxoacids, including 2-oxoisocaproate and 2-oxomethiobutyrate, in addition to pyruvate, oxaloacetate and 2-oxoglutarate. The functionality of the expression system was confirmed by constructing two variants; one (Arg389) being a completely inactive enzyme; the other (Arg283) retaining its full activity, as predicted from the recently solved three-dimensional structure of T. cruzi TAT. Thus, only one of the two strictly conserved arginines which are essential for the enzymatic activity of subfamily Ialpha aspartate and aromatic aminotransferases is critical for T. cruzi's TAT activity.     

The crystal structure of an engineered monomeric triosephosphate isomerase, monoTIM: the correct modelling of an eight-residue loop

BACKGROUND: The triosephosphate isomerase (TIM) fold is found in several different classes of enzymes, most of which are oligomers; TIM itself always functions as a very tight dimer. It has recently been shown that a monomeric form of TIM ('monoTIM') can be constructed by replacing a 15-residue interface loop, loop-3, with an eight-residue fragment; modelling suggests that this should result in a short strain-free turn, resulting in the subsequent helix, helix-A3, having an additional turn at its amino terminus. RESULTS: The crystal structure of monoTIM shows that it retains the characteristic TIM-barrel (betaalpha)8-fold and that the new loop has a structure very close to that predicted. Two other interface loops, loop-1 and loop-4, which contain the active site residues Lys13 and His95, respectively, show significant changes in structure in monoTIM compared with dimeric wild-type TIM. CONCLUSION: The observed structural differences between monoTIM and wild-type TIM indicate that the dimeric appearance of TIM determines the location and conformation of two of the four catalytic residues.     

The crystal structure of triosephosphate isomerase (TIM) from Thermotoga maritima: a comparative thermostability structural analysis of ten different TIM structures

The molecular mechanisms that evolution has been employing to adapt to environmental temperatures are poorly understood. To gain some further insight into this subject we solved the crystal structure of triosephosphate isomerase (TIM) from the hyperthermophilic bacterium Thermotoga maritima (TmTIM). The enzyme is a tetramer, assembled as a dimer of dimers, suggesting that the tetrameric wild-type phosphoglycerate kinase PGK-TIM fusion protein consists of a core of two TIM dimers covalently linked to 4 PGK units. The crystal structure of TmTIM represents the most thermostable TIM presently known in its 3D-structure. It adds to a series of nine known TIM structures from a wide variety of organisms, spanning the range from psychrophiles to hyperthermophiles. Several properties believed to be involved in the adaptation to different temperatures were calculated and compared for all ten structures. No sequence preferences, correlated with thermal stability, were apparent from the amino acid composition or from the analysis of the loops and secondary structure elements of the ten TIMs. A common feature for both psychrophilic and T. maritima TIM is the large number of salt bridges compared with the number found in mesophilic TIMs. In the two thermophilic TIMs, the highest amount of accessible hydrophobic surface is buried during the folding and assembly process.     

Structural and biochemical characterization of a recombinant triosephosphate isomerase from Rhipicephalus (Boophilus) microplus

Triosephosphate isomerase (TIM) is an enzyme with a role in glycolysis and gluconeogenesis by catalyzing the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This enzyme has been used as a target in endoparasite drug development. In this work we cloned, expressed, purified and studied kinetic and structural characteristics of TIM from tick embryos, Rhipicephalus (Boophilus) microplus (BmTIM). The Km and Vmax of the recombinant BmTIM with glyceraldehyde 3-phosphate as substrate, were 0.47 mM and 6031 ??mol min⁻¹ mg protein⁻¹, respectively. The resolution of the diffracted crystal was estimated to be 2.4 Å and the overall data showed that BmTIM is similar to other reported dimeric TIMs. However, we found that, in comparison to other TIMs, BmTIM has the highest content of cysteine residues (nine cysteine residues per monomer). Only two cysteines could make disulfide bonds in monomers of BmTIM. Furthermore, BmTIM was highly sensitive to the action of the thiol reagents dithionitrobenzoic acid and methyl methane thiosulfonate, suggesting that there are five cysteines exposed in each dimer and that these residues could be employed in the development of species-specific inhibitors.     

Stabilization of a reaction intermediate as a catalytic device: definition of the functional role of the flexible loop in triosephosphate isomerase

The function of the mobile loop of triosephosphate isomerase has been investigated by deleting four contiguous residues from the part of this loop that interacts directly with the bound substrate. From the crystal structure of the wild-type enzyme, it appears that this excision will not significantly alter the conformation of the rest of the main chain of the protein. The specific catalytic activity of the purified mutant enzyme is nearly 10(5)-fold lower than that of the wild type. Kinetic measurements and isotopic partitioning studies show that the decrease in activity is due to much higher activation barriers for the enolization of enzyme-bound substrate. Although the substrates bind somewhat more weakly to the mutant enzyme than to the wild type, the intermediate analogue phosphoglycolohydroxamate binds much less well (by 200-fold) to the mutant. It seems that the deleted residues of the loop contribute critically to the stabilization of the enediol phosphate intermediate. Consistent with this view, the mutant enzyme can no longer prevent the loss of the enediol phosphate from the active site and its rapid decomposition to methylglyoxal and inorganic phosphate. Indeed, when glyceraldehyde 3-phosphate is the substrate, the enediol phosphate intermediate is lost (and decomposes) 5.5 times faster than it reprotonates to form the product dihydroxyacetone phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of two new electrophoretic variants of human triosephosphate isomerase: Stability,kinetic, and immunological properties

Two new electrophoretic variants of human triosephosphate isomerase (TPI) have been partially purified and characterized. The TPI Manchester variant, a cathodally migrating electrophoretic allozyme identified in an individual with the phenotype TPI 1-Manchester, is associated with a normal level of enzyme activity in erythrocytes and normal kinetic properties. It is very thermolabile at 55 and 57° C, although it is not uniquely sensitive to either guanidine-HCl or urea denaturation. The TPI Hiroshima-2 variant is an anodally migrating allozyme (the phenotype of proband is TPI 1-Hiroshima-2) with normal activity and kinetic properties and also normal stability characteristics. It is inactivated less by antisera raised against normal human TPI than either the normal or the Manchester allozyme. Dissociation-reassociation experiments utilizing these allozymes have confirmed that normal human red blood cell TPI isozymes are produced by a sequence of reactions (presumably deamidations) involving alternating subunits.Financial support was derived from Contract EY-77-C-02-2828 from the Department of Energy.     

Origin of the triosephosphate isomerase isozymes in humans: genetic evidence for the expression of a single structural locus.

A genetic approach is used to ascertain that a single structural locus for triosephosphate isomerase (TPI) (E.C.5.3.1.1.) is expressed in rapidly dividing human lymphoblasts. This approach is made possible through the identification of a rare electrophoretic variant of human TPI. The variant phenotype is expressed by the TPI-B isozyme in both erythrocytes and peripheral lymphocytes. The variant phenotype is also expressed in the thermostability and electrophoretic pattern of the TPI-A isozyme in mitogen-stimulated lymphoblasts, indicating that TPI-A and TPI-B are products of the same structural locus. These findings are in contrast to the recent conclusions of Yuan et al. based upon structural analysis, suggesting that the TPI-A and TPI-B isozymes are products of distinct structural loci.     

Segmental motion in catalysis: investigation of a hydrogen bond critical for loop closure in the reaction of triosephosphate isomerase.

A residue essential for proper closure of the active-site loop in the reaction catalyzed by triosephosphate isomerase is tyrosine-208, the hydroxyl group of which forms a hydrogen bond with the amide nitrogen of alanine-176, a component of the loop. Both residues are conserved, and mutagenesis of the tyrosine to phenylalanine results in a 2000-fold drop in the catalytic activity (kcat/Km) of the enzyme compared to the wild-type isomerase. The nature of the closure process has been elucidated from both viscosity dependence and primary isotope effects. The reaction catalyzed by the mutant enzyme shows a viscosity dependence using glycerol as the viscosogen. This dependence can be attributed to the rate-limiting motion of the active-site loop between the "open" and the "closed" conformations. Furthermore, a large primary isotope effect is observed with [1-2H]dihydroxyacetone phosphate as substrate [(kcat/Km)H/(kcat/Km)D = 6 +/- 1]. The range of isotopic experiments that were earlier used to delineate the energetics of the wild-type isomerase has provided the free energy profile of the mutant enzyme. Comparison of the energetics of the wild-type and mutant enzymes shows that only the transition states flanking the enediol intermediate have been substantially affected. The results suggest either that loop closure and deprotonation are coupled and occur in the same rate-limiting step or that these two processes happen sequentially but interdependently. This finding is consistent with structural information that indicates that the catalytic base glutamate-165 moves 2 A toward the substrate upon loop closure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence determinants for the reaction specificity of murine (12R)-lipoxygenase: targeted substrate modification and site-directed mutagenesis

Mammalian lipoxygenases (LOXs) are categorized with respect to their positional specificity of arachidonic acid oxygenation. Site-directed mutagenesis identified sequence determinants for the positional specificity of these enzymes, and a critical amino acid for the stereoselectivity was recently discovered. To search for sequence determinants of murine (12R)-LOX, we carried out multiple amino acid sequence alignments and found that Phe(390), Gly(441), Ala(455), and Val(631) align with previously identified positional determinants of S-LOX isoforms. Multiple site-directed mutagenesis studies on Phe(390) and Ala(455) did not induce specific alterations in the reaction specificity, but yielded enzyme species with reduced specific activities and stereo random product patterns. Mutation of Gly(441) to Ala, which caused drastic alterations in the reaction specificity of other LOX isoforms, failed to induce major alterations in the positional specificity of mouse (12R)-LOX, but markedly modified the enantioselectivity of the enzyme. When Val(631), which aligns with the positional determinant Ile(593) of rabbit 15-LOX, was mutated to a less space-filling residue (Ala or Gly), we obtained an enzyme species with augmented catalytic activity and specifically altered reaction characteristics (major formation of chiral (11R)-hydroxyeicosatetraenoic acid methyl ester). The importance of Val(631) for the stereo control of murine (12R)-LOX was confirmed with other substrates such as methyl linoleate and 20-hydroxyeicosatetraenoic acid methyl ester. These data identify Val(631) as the major sequence determinant for the specificity of murine (12R)-LOX. Furthermore, we conclude that substrate fatty acids may adopt different catalytically productive arrangements at the active site of murine (12R)-LOX and that each of these arrangements may lead to the formation of chiral oxygenation products.     

d-ephedrinephosphate,DEP: A new substrate with specificity for prostatic acid phosphatase (PAP)

Summary This report describes the synthesis and physical (including spectral) properties of a new substrate, d-ephedrinephosphate, DEP¹, which is histochemically highly specific for a secreted non-lysosomal prostatic acid phosphatase, PAP. This specificity is in contrast to other substrates which are nonspecific, i.e., which demonstrate acid phosphatases that originate from various cell types and are mainly lysosomal. When this substrate is used for light and electron microscopic histochemistry in a modified Gomori medium, PAP is demonstrated mainly in secretory granules and in the Golgi apparatus (and its related vacuoles) of prostatic epithelial cells of several species of mammals including man. This corroborates our previous suggestion that PAP is not a lysosomal enzyme as are many of the other acid phosphatases. This high degree of specificity of DEP for PAP supports the usefulness of this compound in the histochemical and biochemical characterization of PAP, and in the diagnosis of localized or disseminated prostatic disease.This investigation was supported by research grants CA-02478 from the National Cancer Institute, NIH, U.S. Public Health Service, Bethesda, MD and CA-16077 the National Prostatic Cancer Project, NCI, Buffalo, NYDedicated to the memory of the late Arnold M. Seligman, M.D., American Cancer Society Professor of Research Oncology and Cell Biology, who pioneered the development of modern enzyme cytochemistry and under whose direction part of this work was initiated     

Nucleotide variation in the triosephosphate isomerase (Tpi) locus of Drosophila melanogaster and Drosophila simulans

DNA sequence variation in a 1.1-kb region including the coding portion of the Tpi locus was examined in 25 homozygous third-chromosome lines of Drosophila melanogaster, nine lines of Drosophila simulans, and one line of Drosophila yakuba. Our data show that the widespread allozyme polymorphism observed in cosmopolitan D. melanogaster is due to a glutamic acid substitution occurring in a phylogenetically conserved lysine that has been identified as part of the "hinged-lid" active site of the enzyme. This observation suggests that the replacement polymorphism may have important functional consequences. One replacement polymorphism was also observed in D. simulans, although its functional relevance is more difficult to assess, since it affects a site that is not strongly conserved. This amino acid change in D. simulans is associated with a single lineage possessing seven unique silent substitutions, which may be indicative of balancing selection or population subdivision. The absence of fixed amino acid differences between D. melanogaster and D. simulans and only a single difference with D. yakuba suggests that triose phosphate isomerase is under strong functional constraint. Silent variation is slightly higher for D. melanogaster than for D. simulans. Finally, we outline the general lack of evidence for old balanced polymorphisms at allozyme loci in D. melanogaster.