Chicken triosephosphate isomerase (TIM) has been fragmented by inserting single "splits" at three separate exon/exon boundaries, and the complexes have been assayed for catalytic activity. In vivo studies show that the expression of both portions of each of the three different split genes complements the TIM deficiency of Escherichia coli strain DF502 when grown on selective media. The expression of only one fragment of each split gene does not complement the TIM-minus genotype. To assess the catalytic activity that derives from the fragmented protein complex, the individual peptide products of one of the three split genes were expressed and purified. The purified complex showed isomerase activity, albeit of low specific catalytic activity. A catalytically active multichain complex composed of separate peptide products of a gene singly split at exon/exon junctions has thus been created.

The active site and mechanism of action of the enzyme phosphoenolpyruvate mutase have been probed using substrate and intermediate analogues as inhibitors of the mutase-catalyzed reaction. Smaller anions (e.g. sulfite, nitrate, phosphinate, and bicarbonate) are noncompetitive inhibitors of the mutase, while larger anions in the complementary series (sulfate, phosphonate, phosphate) inhibit competitively. Combining oxalate, an intermediate analogue that is a potent inhibitor of the mutase (Ki = 25 microM), with small, noncompetitive inhibitor anions results in synergistic inhibition of the mutase, suggesting that the combined presence of oxalate and anion creates a "bimolecular transition-state analogue". The phosphoenolpyruvate (PEP) mutase genes from Tetrahymena and Streptomyces are known, and these enzymes share significant amino acid sequence similarity to the isocitrate lyase gene from Ricinus. Despite their seeming structural unrelatedness to the substrates of PEP mutase, several isocitrate analogues are good inhibitors, suggesting that isocitrate lyase and PEP mutase are evolutionarily related. An active-site model has been developed that is in accord with the data presented, which are consistent with a mechanism involving the intermediacy of a phosphoenzyme.

The crystal structure of recombinant chicken triosephosphate isomerase (TIM, E.C. 5.3.1.1) complexed with the intermediate analogue phosphoglycolohydroxamate (PGH) has been solved by the method of molecular replacement and refined to an R-factor of 18.5% at 1.8-A resolution. The structure is essentially identical to that of the yeast TIM-PGH complex [Davenport, R. C., et al. (1991) Biochemistry 30, 5821-5826] determined earlier and refined at comparable resolution. This identity extends to the high-energy conformations of the active-site residues Lys13 and Ser211, as well as the positions of several bound water molecules that are retained in the active site when PGH is bound. Comparison with the structure of uncomplexed chicken TIM shows that the catalytic base, Glu165, moves several angstroms when PGH binds. This movement may provide a trigger for a larger conformational change, one of 7 A, in a loop near the active site, which folds down like a lid to shield the bound inhibitor and catalytic residues from contact with bulk solvent. These same conformational changes were seen in crystalline yeast TIM upon binding of PGH; their occurrence here in a different crystal form of TIM eliminates the possibility that they are an artifact of crystal packing.

The Fourier transform infrared (FTIR) spectrum of the complex between prephenate and the monofunctional chorismate mutase from Bacillus subtilis displays one prominent band at 1714 cm(-1). Using isotopically-labeled ligand, we have shown that this band corresponds to the ketonic carbonyl stretching vibration of enzyme-bound prephenate. The frequency of this carbonyl vibration of prephenate does not change significantly on binding to the protein. These data indicate that chorismate mutase does not use electrophilic catalysis in the rearrangement of chorismate. A comparison of the resolution-enhanced FTIR spectra of the unliganded mutase and of the protein complexed with its ligands reveals marked differences in the amide I' vibration band. These changes suggest that structural alterations in the protein occur upon binding prephenate. When combined with information from the crystal structure of the enzyme and its complexes, it appears that significant ordering of the C-terminal region occurs upon ligand binding. These changes at the active site may be important for efficient catalysis and likely influence the association and dissociation rates of the enzyme and its ligands. The enzymic rearrangement of chorismate evidently proceeds via a pericyclic process, and much, if not all, of the rate acceleration derives from the selective binding of the appropriate conformer of the substrate, with some additional contribution possible from electrostatic stabilization of the transition state.

Enolase catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate. Kinetic isotope effects have been used to determine whether abstraction of the proton from C-2 and loss of hydroxide from C-3 of 2-phosphoglycerate occur in a concerted reaction or as sequential processes and whether these steps are kinetically significant for the enolase-catalyzed reaction. Enolase exhibits a significant primary deuterium isotope effect, as well as catalyzing the relatively rapid exchange of the C-2 proton with solvent water. Secondary C-3 deuterium isotope effects are also reported, both when the C-2 carbon carries a hydrogen and when this center is deuterated. These results provide information about the kinetic significance and timing of the transition state(s) associated with the loss of H+ and OH-. Strong evidence has been presented for a stepwise mechanism where both the rate of proton abstraction and one or both of the later transition states, i.e., those associated with hydroxide loss and product release, limit the overall reaction rate. If a concerted reaction were to be invoked, the presence of a small secondary 2H isotope effect in combination with the observed rate of exchange of the C-2 proton require the intrinsic secondary 2H kinetic isotope effect to be effectively unity. For the concerted mechanism, an intrinsic effect of unity would be consistent only with an extremely asymmetric transition state that is dominated by C-H bond cleavage.

The role of lysine-12 at the active site of yeast triosephosphate isomerase has been elucidated by a combination of site-directed mutagenesis, Fourier transform infrared spectroscopy, enzyme kinetics, and X-ray crystallography. Several lines of evidence suggest that the mutant isomerase in which lysine has been changed to methionine cannot bind substrate. This mutant enzyme has no detectable catalytic activity, and infrared experiments show no evidence of binding dihydroxyacetone phosphate nor dihydroxyacetone sulfate to the active site. Furthermore, crystals of the enzyme grown in the presence of phosphoglycolohydroxamate, a potent reaction intermediate analog, show an open active site with no inhibitor bound. Mutation of lysine-12 to arginine produces a protein with a value Km elevated by a factor of 22, a Vmax reduced by a factor of 180, and a Ki for phosphoglycolohydroxamate elevated by a factor of 290. Mutation of lysine-12 to histidine produces an enzyme that shows virtually no catalytic activity at neutral pH, but below pH 6.1 this enzyme is active, suggesting that protonation of the histidine in this mutant is required for activity. These studies, together with the structural results reported in an accompanying paper, provide convincing evidence that a positive charge is required for substrate binding at the active site of triosephosphate isomerase and that lysine-12 provides this positive charge.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTPurple acid phosphatase: a diiron enzyme that catalyzes a direct phospho group transfer to waterEugene G. Mueller, Michael W. Crowder, Bruce A. Averill, and Jeremy R. KnowlesCite this: J. Am. Chem. Soc. 1993, 115, 7, 2974–2975Publication Date (Print):April 1, 1993Publication History Published online1 May 2002Published inissue 1 April 1993https://doi.org/10.1021/ja00060a055Request reuse permissionsArticle Views328Altmetric-Citations88LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (216 KB) Get e-AlertscloseSupporting Info (2)»Supporting Information Supporting Information Get e-Alerts

In previous work, we have shown that the first (and, presumably, the second) pK(a) of the active-site histidine-95 in triosephosphate isomerase has been lowered by about 2 units [Lodi, P. J., & Knowles, J. R. (1991) Biochemistry 30, 6948-6956]. One reason for the perturbed pK(a) of this residue appears to be its location at the N-terminus of a short alpha-helix that runs from residues 95 to 102. Fortuitously, the residue at the C-terminus of this helix is also a histidine residue (histidine-103), and the existence of a histidine side chain at each end has allowed us directly to implicate the helix in the perturbation of the pK(a) value of histidine-95. N-15 NMR titration studies of the native enzyme and C-13 NMR titration studies of the denatured enzyme show that while the pK(a) of histidine-95 is lowered by a least 2 units in the folded versus the unfolded state, the pK(a) of histidine-103 is raised by about 0.6 unit on protein folding. These complementary effects on the pK(a) values of histidine-95 and histidine-103 suggest that the alpha-helix is indeed responsible for the perturbation of the pK(a) values. The larger effect on the pK(a) of histidine-95 is readily rationalized in terms of the local structure of the enzyme. The disparity in the perturbation for the two histidine side chains illustrates how an alpha-helix can be functionally utilized by proteins, directly to affect (as in the present case) the chemistry of catalysis by an enzyme.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Primary kinetic isotope effects on Vmax were observed in both reaction directions upon racemizing samples of [2-2H]glutamate with the cofactor-independent glutamate racemase from Lactobacillus. This supports a deprotonation/protonation mechanism for racemization in which the breaking of the carbon-hydrogen bond at C-2 is partially rate-determining. Substantial "overshoots" were observed when the time course of racemization of either enantiomer of glutamate was monitored using circular dichroism spectroscopy. This is consistent with a "two-base" mechanism accompanied by a kinetic isotope effect. "Competitive deuterium washout" experiments were used to measure kinetic isotope effects on Vmax/Km of 2.5 for (S)-glutamate and 3.4 for (R)-glutamate. The ratio of the notably different isotope effects was confirmed by "double competitive deuterium washout" experiments. Site-directed mutagenesis was used to generate the mutant C73A and C184A enzymes. In each case the mutant enzymes were inactive as racemases. The two mutant enzymes are, however, capable of catalyzing the elimination of HCl from opposite enantiomers of threo-3-chloroglutamic acid, a process that presumably requires only one enzymic base. This finding indicates that the active sites of the mutant enzymes are intact and that the two cysteines flank the bound substrate molecule. It appears that cysteine-73 is responsible for the abstraction of the C-2 hydrogen from (R)-glutamate and cysteine-184 abstracts the proton from (S)-glutamate in the racemization reaction of the wild-type enzyme.

Glutamate racemase has been purified more than 12,000-fold from Lactobacillus fermenti. The racemase gene has been cloned using standard hybridization techniques combined with a novel selection for in vivo glutamate racemase activity, and the racemase has been expressed in Escherichia coli as 20-25% of the total soluble cell protein. The cloned gene product is indistinguishable from that purified from Lactobacillus and is a monomer of M(r) 28,300. Both a coupled enzymatic assay and a circular dichroism assay show that the enzyme follows Michaelis-Menten kinetics, with a Km of 0.3 mM and a kcat of 70 s-1 in each reaction direction. Investigations into the cofactor dependence of glutamate racemase indicate that the enzyme employs neither pyridoxal phosphate nor a pyruvoyl group in the labilization of the proton at the stereogenic center of glutamate. Furthermore, the racemase activity is unaffected by the presence of the metal-chelating reagent EDTA. The gene sequence of the racemase is 24% identical to that of aspartate racemase from Streptococcus thermophilus and 30% identical to that of an unidentified open reading frame in the rrnB ribosomal RNA operon of E. coli. Because the two cysteine residues in glutamate racemase and their surrounding regions are well-conserved in both of these sequences, and since glutamate racemase is stabilized by the presence of reduced thiols, these residues are possible candidates for the enzymic bases that deprotonate glutamate at C-2.

The simplest chemical transformations in metabolism are the proton transfer reactions exemplified by certain isomerases and racemases. We have been studying three such enzymes to understand the structural features that lead to efficient proton transfer. All of these enzymes face the common problem of abstracting a hydrogen from a carbon acid of high pKa with an enzymic base of low pKa. We have used X-ray crystallography, site-directed mutagenesis, and molecular dynamics simulations to arrive at a set of principles for optimal catalysis of this simple reaction.

The mechanism of the cofactor-independent glutamate racemase from Lactobacillus has been studied. The possible formation of an acylenzyme intermediate during catalysis has been investigated using 18O-carboxyl labeled glutamate. The absence of any washout of label during racemization argues against intermediate formation. The observation of the enzyme-catalyzed incorporation of deuterium at the C-2 position of glutamate upon racemization in D2O provides evidence for a deprotonation/protonation mechanism. Further experiments have been performed in order to determine the number of enzymic bases responsible for racemization. Solvent deuterium is efficiently incorporated into the product enantiomer but not into the recovered substrate enantiomer in each reaction direction. This finding is consistent with a "two-base" mechanism in which one enzymic base deprotonates the substrate, and the conjugate acid of a second enzymic base protonates the resulting intermediate from the opposite face. It also suggests that the two bases are monoprotic. The possibility that the two enzymic forms, which differ at the very least by the protonation states of the active-site bases, are kinetically significant has been examined by measuring the entire time course of the approach to equilibrium at various concentrations of glutamate. An "oversaturated" regime [Fisher, L. M., Albery, W. J., & Knowles, J. R. (1986) Biochemistry 25, 2529-2537] was not observed using glutamate concentrations as high as 100 mM, indicating that the two enzyme forms are rapidly interconverting under physiological conditions.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

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)

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.

Attachment of influenza virus to susceptible cells is mediated by the viral protein hemagglutinin, which recognizes cell-membrane-bound glycoconjugates that terminate in alpha-sialosides. We have synthesized a fluorescent alpha-sialoside that has the highest affinity of any reported monovalent ligand for hemagglutinin, and it is not a substrate for the viral neuraminidase. This alpha-sialoside provides a convenient fluorescence competition assay for the binding of other ligands. Since each of the currently used binding assays has significant disadvantages, such a simple assay is of great importance for the study of potential inhibitors of viral attachment.

The phosphoenolpyruvate mutase gene from Tetrahymena pyriformis has been cloned and overexpressed in Escherichia coli. To our knowledge, this is the first Tetrahymena gene to be expressed in E. coli, a task made more complicated by the idiosyncratic codon usage by Tetrahymena. The N-terminal amino acid sequence of phosphoenolpyruvate mutase purified from T. pyriformis has been used to generate a precise oligonucleotide probe for the gene, using in vitro amplification from total genomic DNA by the polymerase chain reaction. Use of this precise probe and oligo(T) as primers for in vitro amplification from a T. pyriformis cDNA library has allowed the cloning of the mutase gene. A similar amplification strategy from genomic DNA yielded the genomic sequence, which contains three introns. The sequence of the DNA that encodes 10 amino acids upstream of the N-terminal sequence of the isolated protein was found by oligonucleotide hybridization to a subgenomic library. These 10 N-terminal amino acids are cleanly removed in Tetrahymena in vivo. The full mutase gene sequence codes for a protein of 300 amino acids, and it includes two amber (TAG) codons in the open reading frame. In Tetrahymena, TAG codes for glutamine. When the two amber codons are each changed to a glutamine codon (CAG) that is recognized by E. coli and the gene is placed behind a promoter driven by the T7 RNA polymerase, expression in E. coli is observed. The mutase gene also contains a large number of arginine AGA codons, a codon that is very rarely used by E. coli. Cotransformation with a plasmid carrying the dnaY gene [which encodes tRNA(Arg)(AGA)] results in more than 4-fold higher expression. The mutase then comprises about 25% of the total soluble cell protein in E. coli transformants. The mutase gene bears significant similarity to one other gene in the available data bases, that of carboxyphosphonoenolpyruvate mutase from Streptomyces hygroscopicus, an enzyme that catalyzes a closely related transformation. Due to the large evolutionary distance between Tetrahymena and Streptomyces, this similarity can be interpreted as the first persuasive evidence that the biosynthesis of phosphonates is an ancient metabolic process.

To illuminate the role of histidine-95 in the catalytic reaction mediated by triosephosphate isomerase, 13C and 15N NMR titration studies have been carried out both on the wild-type enzyme and on a mutant isomerase in which the single remaining histidine (that at the active site) has been isotopically enriched in the imidazole ring. 15N NMR has proved especially useful in the unambiguous demonstration that the imidazole ring of histidine-95 is uncharged over the entire pH range of isomerase activity, between pH 5 and pH 9.9. The results require that the first pKa of histidine-95 is below 4.5. This abnormally low pKa rules out the traditional view that the positively charged imidazolium cation of histidine-95 donates a proton to the developing charge on the substrate's carbonyl oxygen. 15N NMR experiments on the enzyme in the presence of the reaction intermediate analogue phosphoglycolohydroxamate show the presence of a strong hydrogen bond between N epsilon 2 of histidine-95 and the bound inhibitor. These findings indicate that, in the catalyzed reaction, proton abstraction from C-1 of dihydroxyacetone phosphate first yields an enediolate intermediate that is strongly hydrogen bonded to the neutral imidazole side chain of histidine-95. The imidazole proton involved in this hydrogen bond then protonates the enediolate, with the transient formation of the enediol-imidazolate ion pair. Abstraction of the hydroxyl proton on O-1 now produces the other enediolate intermediate, which collapses to give the product glyceraldehyde 3-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)