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PubMed2001

Superefficient enzymes.

Diffusion-controlled enzymes are characterized by second-order rate constants in the range 10(8)-10(10) M(-1)s(-1). These values are at the upper end of the observed rates of many enzyme-substrate reactions and have been predicted by theoretical studies on bimolecular reaction in solution. Such enzymes are considered to be perfect, since their rate-limiting step is not due to any chemical event but to the diffusional association rate between the enzyme and the substrate. Often the enzyme-substrate encounter is facilitated either through the presence of a strong attractive electric field, produced by charges on the enzyme surface, or through the reduction in the dimension of the search process. Here we provide a brief review of some of the enzymes characterized by a very fast second-order constant, focusing attention on triose phosphate isomerase and Cu,Zn superoxide dismutase taken as typical examples of such highly tuned enzymes.

Animals↗
PubMed1999

Monophyly of lampreys and hagfishes supported by nuclear DNA-coded genes.

The phylogenetic position of hagfishes in vertebrate evolution is currently controversial. The 18S and 28S rRNA trees support the monophyly of hagfishes and lampreys. In contrast, the mitochondrial DNAs suggest the close association of lampreys and gnathostomes. To clarify this controversial issue, we have conducted cloning and sequencing of the four nuclear DNA-coded single-copy genes encoding the triose phosphate isomerase, calreticulin, and the largest subunit of RNA polymerase II and III. Based on these proteins, together with the Mn superoxide dismutase for which hagfish and lamprey sequences are available in database, phylogenetic trees have been inferred by the maximum likelihood (ML) method of protein phylogeny. It was shown that all the five proteins prefer the monophyletic tree of cyclostomes, and the total log-likelihood of the five proteins significantly supports the cyclostome monophyly at the level of +/-1 SE. The ML trees of aldolase family comprising three nonallelic isoforms and the complement component group comprising C3, C4, and C5, both of which diverged during vertebrate evolution by gene duplications, also suggest the cyclostome monophyly.

Animals↗
PubMed1992

A TIM barrel protein without enzymatic activity? Crystal-structure of narbonin at 1.8 A resolution.

The major protein component in seeds is storage protein. These have no known enzymatic activity and act to provide amino acids as a source of metabolites in the developing seedling. We report here the first three dimensional crystal structure of a seed storage globulin at high resolution. The molecule of the 2S globulin, narbonin, from Vicia narbonensis L., consists of an eight-stranded parallel alpha/beta barrel structure similar to that observed in triose phosphate isomerase (TIM). Narbonin is the first protein with this topology possessing no known enzymatic activity. Because of the lack of sequence information most of the primary structure was determined directly from the electron density.

Amino Acid Sequence↗
PubMed1986

Size-fractionation of the small chromosomes of Trypanozoon and Nannomonas trypanosomes by pulsed field gradient gel electrophoresis.

We have compared the molecular karyotypes of trypanosomes from different subgroups within subgenus Trypanozoon by pulsed field gradient (PFG) gel electrophoresis. Although the overall karyotype was similar, there was much variation in the size of chromosomes between different stocks. Two of three stocks of Trypanosoma (Trypanozoon) brucei gambiense had remarkably small mini-chromosomes: 25-50 kilobase pairs compared to 50-150 kilobase pairs for the mini-chromosomes of other Trypanozoon stocks. The relative amount of DNA in the mini-chromosomal fraction of different stocks correlated well with the amount of 177 base pair satellite DNA monomer per microgram nuclear DNA. Hybridisation of Southern blots of pulsed field gradient gels with a number of gene probes showed that the loci for tubulin and phosphoglycerate kinase in Trypanozoon probably lie on the same chromosome, together with some variant surface glycoprotein genes; the genes for triose phosphate isomerase and glyceraldehyde phosphate dehydrogenase are separately located both with respect to each other and the above housekeeping genes. Therefore, there are at minimum three pairs of chromosomes carrying housekeeping genes in Trypanozoon. In some stocks the chromosomes carrying the tubulin and phosphoglycerate kinase genes are split into two bands, suggesting that homologous chromosomes may differ substantially in size in trypanosomes. One Trypanosoma (Nannomonas) congolense stock examined had a similar pattern of chromosome distribution to that of Trypanozoon, but with very small mini-chromosomes (25-50 kilobase pairs.)

Animals↗
PubMed1989

Glycolytic enzyme interactions with tubulin and microtubules.

Interactions of the glycolytic enzymes glucose-6-phosphate isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, triose-phosphate isomerase, enolase, phosphoglycerate mutase, phosphoglycerate kinase, pyruvate kinase, lactate dehydrogenase type-M, and lactate dehydrogenase type-H with tubulin and microtubules were studied. Lactate dehydrogenase type-M, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and aldolase demonstrated the greatest amount of co-pelleting with microtubules. The presence of 7% poly(ethylene glycol) increased co-pelleting of the latter four enzymes and two other enzymes, glucose-6-phosphate isomerase, and phosphoglycerate kinase with microtubules. Interactions also were characterized by fluorescence anisotropy. Since the KD values of glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase for tubulin and microtubules were all found to be between 1 and 4 microM, which is in the range of enzyme concentration in cells, these enzymes are probably bound to microtubules in vivo. These observations indicate that interactions of cytosolic proteins, such as the glycolytic enzymes, with cytoskeletal components, such as microtubules, may play a structural role in the formation of the microtrabecular lattice.

Animals↗
PubMed1995

Inhibition of glycolysis in boar spermatozoa by 1,6-dichloro-1,6-dideoxy-D-fructose.

The dichloro-analogue of D-fructose 1,6-bisphosphate, 1,6-dichloro-1,6-dideoxy-D-fructose, is a weak substrate for boar sperm aldolase which converts it to (S)-3-chlorolactaldehyde and 3-chloro-1-hydroxypropanone in vitro. Production of these chloro-trioses leads to the strong inhibition of glyceraldehyde-3-phosphate dehydrogenase, the weak inhibition of triosephosphate isomerase and the transient inhibition of aldolase.

Animals↗
PubMed2006

Identification and characterization of triosephosphate isomerase that specifically interacts with the integrin alphaIIb cytoplasmic domain.

Integrin alphaIIb/beta3 (IIb/IIIa), a platelet fibrinogen receptor, has been shown to play a critical role in thrombosis and hemostasis. However, the mechanisms by which ligands interact with the alphaIIb/beta3 receptor is not very clear at this time. The interaction between the ligand, the receptor and the transmission of extracellular signals may involve the cytoplasmic domains of these integrins. The objective of this investigation was to identify novel proteins that interact with the cytoplasmic tail of alphaIIb. Using alphaIIb cytoplasmic tail as the bait and a yeast two-hybrid system, we have identified three separate clones containing inserts that encoded the same protein with different truncated N-terminals. Sequence analysis showed that the inserts of the three clones encoded a previously identified enzyme: triose phosphate isomerase (TPI). In addition, we demonstrated that TPI failed to interact with the integrin alpha2 tail, beta3 tail and lamin, but showed a weak binding to the alphaV tail which shares the highest homology with alphaIIb tail among the integrin alpha family. Site-directed mutagenesis studies around the homology region indicated that the critical peptide sequence necessary for the interaction between TPI and alphaIIb tail is GFFKRNRPPLEE. Using RT-PCR, we have demonstrated the presence of TPI mRNA in platelets. In addition, experiments were also performed to demonstrate specific binding of TPI to alphaIIb using an ELISA and fusion protein. Taken together, these data suggest that TPI specifically interacts with alphaIIb and may play a critical role in alphaIIb/beta3-mediated platelet function.

Amino Acid Sequence↗
PubMed2006

Local encoding of computationally designed enzyme activity.

One aim of computational protein design is to introduce novel enzyme activity into proteins of known structure by predicting mutations that stabilize transition states. Previously, we showed that it is possible to introduce triose phosphate isomerase activity into the ribose-binding protein of Escherichia coli by constructing 17 mutations in the first two layers of residues that surround the wild-type ligand-binding site. Here, we report that these mutations can be "transplanted" into a homologous ribose-binding protein, isolated from the hyperthermophilic bacterium Thermoanaerobacter tengcongensis, with retention of catalytic activity, substrate affinity, and reaction pH dependence. The observed 10(5)-10(6)-fold rate enhancement corresponds to 70% of the maximally known transition-state binding energy. The wild-type sequences in these two homologues are almost perfectly conserved in the vicinity of their ribose-binding sites, but diverge significantly at increasing distance from these sites. The results demonstrate that the computationally designed mutations are sufficient to encode the observed enzyme activity, that all the observed activity is encoded locally within the layer of residues directly in contact with the substrate and that, in this case, at least 70% of transition state stabilization energy can be achieved using straightforward considerations of stereochemical complementarity between enzyme and reactants.

Amino Acid Sequence↗
PubMed1986

Pyrophosphate-dependent sucrose metabolism and its activation by fructose 2,6-bisphosphate in sucrose importing plant tissues.

In the presence of pyrophosphate and uridine diphosphate, sucrose was cleaved to form glucose 1-phosphate and fructose with soluble extracts from sucrose importing plant tissues. The glucose 1-phosphate then was converted through glycolysis to triose phosphates in a pyrophosphate-dependent pathway which was activated by fructose 2,6-bisphosphate. Much less activity, less than 5%, was found in sucrose exporting tissue extracts from the same plants. These findings suggest that imported sucrose is metabolized in the cytoplasm of plant tissues by utilizing pyrophosphate and that sucrose metabolism is partially regulated by fructose 2,6-bisphosphate.

Biological Transport↗
PubMed2002

Iterative database searches demonstrate that glycoside hydrolase families 27, 31, 36 and 66 share a common evolutionary origin with family 13.

Classification of glycoside hydrolases (GHs) into families, along with the structure-based grouping together of families into clans, improve our understanding of the evolution of the large natural variety of these enzymes, help rationalise experimental data and guide further studies. Here we identify triose phosphate isomerase (TIM) barrels in GH families 27, 31, 36 and 66. We further show that iterated sequence database searches provide evidence for their sharing a common evolutionary origin with GH family 13. The catalytic, nucleophilic residue common to all these families is thereby determined and candidate catalytic proton donors identified within each family.

Aspartic Acid↗
PubMed1998

Molecular comparison of Giardia lamblia isolates.

Giardia lamblia (also Giardia duodenalis, Giardia intestinalis) isolates have been variably divided into two or three genotypes by different investigators. We have compared the triose phosphate isomerase sequences of the three genotypes (Groups 1, 2, and 3) described by Nash and shown that Groups 1 and 2 are similar, while Group 3 is markedly different from Groups 1 and 2, indicating that Group 1/2 and Group 3 correspond to the two major genotypes identified by other investigators. We have also analysed three Chinese isolates and showed that two fit into Group 3, while the third contained a mixture of Groups 1 and 3 isolates. These results confirm the relatedness of G. lamblia isolates from throughout the world, and established the feasibility of using DNA amplification and sequence analysis for detecting mixed isolates.

Amino Acid Sequence↗
PubMed2000

Strategies for metabolic flux analysis in plants using isotope labelling.

Flux measurements through metabolic pathways generate insights into the integration of metabolism, and there is increasing interest in using such measurements to quantify the metabolic effects of mutation and genetic manipulation. Isotope labelling provides a powerful approach for measuring metabolic fluxes, and it gives rise to several distinct methods based on either dynamic or steady-state experiments. We discuss the application of these methods to photosynthetic and non-photosynthetic plant tissues, and we illustrate the different approaches with an analysis of the pathways interconverting hexose phosphates and triose phosphates. The complicating effects of the pentose phosphate pathway and the problems arising from the extensive compartmentation of plant cell metabolism are considered. The non-trivial nature of the analysis is emphasised by reference to invalid deductions in earlier work. It is concluded that steady-state isotopic labelling experiments can provide important information on the fluxes through primary metabolism in plants, and that the combination of stable isotope labelling with detection by nuclear magnetic resonance is particularly informative.

Biotechnology↗
PubMed2004

Epidemiological and molecular evidence supports the zoonotic transmission of Giardia among humans and dogs living in the same community.

Giardia duodenalis isolates recovered from humans and dogs living in the same locality in a remote tea-growing community of northeast India were characterized at 3 different loci; the SSU-rDNA, elongation factor 1-alpha (ef1-alpha) and triose phosphate isomerase (tpi) gene. Phylogenetic analysis of the SSU-rDNA and efl-alpha genes provided poor genetic resolution of the isolates within various assemblages, stressing the importance of using multiple loci when inferring genotypes to Giardia. Analysis of the tpi gene provided better genetic resolution and placed canine Giardia isolates within the genetic groupings of human isolates (Assemblages A and B). Further evidence for zoonotic transmission was supported by epidemiological data showing a highly significant association between the prevalence of Giardia in humans and presence of a Giardia-positive dog in the same household (odds ratio 3.01, 95% CI, 1.11, 8.39, P = 0.0000).

Adolescent↗
PubMed1990

Proton diffusion in the active site of triosephosphate isomerase.

The current model for hydrogen flow in the aldose-ketose isomerases is probably incorrect. Enzymes of this class are characterized by both hydrogen transfer and proton exchange in the interconversion of substrate and product. The transfer is believed to be due to the action of a unique basic residue in the active site. Exchange is presumed to occur by dissociation of the abstracted proton and reassociation from the medium prior to its transfer to the intermediate enediol on the way to product. Dissociation of a necessary proton from the intermediate state imposes limits on the overall catalytic rate depending on the pKa of the protonated base and the pH of the medium. A case in point is triose-P isomerase (TIM), where kcat is approximately 10(4) s-1. T-Labeled substrate is found to lose approximately 95% of its T to the medium when totally converted to product. Although the active site base is believed to be a glutamate of pKa = 3.9, the pH dependence of maximum velocity is known to be flat up to pH 10. The loss of hydrogen required to form product as indicated by isotope exchange must be restored completely at this high pH, requiring a base of very high pKa, or there must be some other explanation for the loss of isotope. The present study demonstrates the existence of a single proton on human and rabbit TIM and three protons on yeast TIM that rapidly exchange with the abstracted proton at the E.enediol state internal exchange. Exchange with the medium external exchange occurs from the enzyme after substrate or product has dissociated.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1976

Altered enzyme expression in "differentiated" murine neuroblastoma cells.

Out of 17 enzymes studied, only 9 were detectable by starch gel electrophoresis in mouse neuroblastoma cells in culture. Prostaglandin E1 (PGE1) and 4(-3-butoxy-4-methoxybenzyl)-2-imidazolidinone (R020-1724), a specific inhibitor of cAMP phosphodiesterase, were used to induce "differentiation". Lactate and 6-phosphogluconate dehydrogenases and adenylate kinase were expressed as single bands in untreated neuroblastoma and induced "differentiated" cells, but the electrophoretic mobility of these enzymes in PGE1-treated cells was slower than that in malignant and R020-1724-treated cells. Three bands of glucose 6-phosphate dehydrogenase were detectable in PGE1-treated cells, whereas the R020-1724-treated cells had two bands and the untreated neuroblastoma cells had only one band. Aldolase was also expressed as a single band; however, the activity of this enzyme was much higher in PGE1-treated cells, whereas the activity was bately detectable for R020-1724-treated and untreated neuroblastoma cells. Some of the enzymes which are present in vivo are absent in vitro. Alkaline phosphatase is present in brain but is absent in neuroblastoma cells in vivo and in vitro. Two bands each of triose phsophate isomerase, fumarase and aldolase are present in brain, but only one band of these enzymes is present in neuroblastoma cells. Although PGE1 and R020-1724 induce many differentiated functions in neuroblastoma cells in a similar manner, PGE1 appears to change characteristically the expression of several enzymes.

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗
PubMed1994

Simulation of enzyme-substrate encounter with gated active sites.

We describe a brownian dynamics simulation method that allows investigation of the effects of receptor flexibility on ligand binding rates. The method is applied to the encounter of substrate, glyceraldehyde 3-phosphate, with triose phosphate isomerase, a diffusion-controlled enzyme with flexible peptide loops at its active sites. The simulations show that while the electrostatic field surrounding the enzyme steers the substrate into its active sites, the flexible loops appear to have little influence on the substrate binding rate. The dynamics of the loops may therefore have been optimized during evolution to minimize their interference with the substrate's access to the active sites. The calculated and experimental rate constants are in good agreement.

Animals↗
PubMed1970

Regulation of lipogenesis. Stimulation of fatty acid synthesis in vivo and in vitro in the liver of the newly hatched chick.

1. A single glucose meal stimulated the incorporation of acetate into fatty acids in liver slices. If the glucose was added in vitro, it had no effect. Fructose and glycerol in vitro markedly stimulated fatty acid synthesis from acetate. Fructose and glycerol probably by-passed a rate-controlling reaction between glucose and triose phosphate. This reaction may have been stimulated by glucose administered in vivo. 2. The stimulation of fatty acid synthesis caused by fructose did not require the synthesis of enzyme, thus indicating that fatty acid-synthesizing enzymes were present in a latent form in the livers from unfed chicks.

Acetates↗
PubMed1970

The regulation of triglyceride synthesis and fatty acid synthesis in rat epididymal adipose tissue. Effects of altered dietary and hormonal conditions.

1. Epididymal adipose tissues obtained from rats that had been previously starved, starved and refed a high fat diet for 72h, starved and refed bread for 144h or fed a normal diet were incubated in the presence of insulin+glucose or insulin+glucose+acetate. 2. Measurements were made of the whole-tissue concentrations of hexose phosphates, triose phosphates, glycerol 1-phosphate, 3-phosphoglycerate, 6-phosphogluconate, adenine nucleotides, acid-soluble CoA, long-chain fatty acyl-CoA, malate and citrate after 1h of incubation. The release of lactate, pyruvate and glycerol into the incubation medium during this period was also determined. 3. The rates of metabolism of glucose in the hexose monophosphate pathway, the glycolytic pathway, the citric acid cycle and into glyceride glycerol, fatty acids and lactate+pyruvate were also determined over a 2h period in similarly treated tissues. The metabolism of acetate to CO(2) and fatty acids in the presence of glucose was also measured. 4. The activities of acetyl-CoA carboxylase, fatty acid synthetase and isocitrate dehydrogenase were determined in adipose tissues from starved, starved and fat-refed, and alloxan-diabetic animals and also in tissues from animals that had been starved and refed bread for up to 96h. Changes in these activities were compared with the ability of similar tissues to incorporate [(14)C]glucose into fatty acids in vitro. 5. The activities of acetyl-CoA carboxylase and fatty acid synthetase roughly paralleled the ability of tissues to incorporate glucose into fatty acids. 6. Rates of triglyceride synthesis and fatty acid synthesis could not be correlated with tissue concentrations of long-chain fatty acyl-CoA, citrate or glycerol 1-phosphate. In some cases changes in phosphofructokinase flux rates could be correlated with changes in citrate concentration. 7. The main lesion in fatty acid synthesis in tissues from starved, starved and fat-refed, and alloxan-diabetic rats appeared to reside at the level of pyruvate utilization and to be related to the rate of endogenous lipolysis. 8. It is suggested that pyruvate utilization by the tissue may be regulated by the metabolism of fatty acids within the tissue. The significance of this in directing glucose utilization away from fatty acid synthesis and into glyceride-glycerol synthesis is discussed.

Acetates↗