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PubMed1996

Cytotoxicity studies of quercetin, shikimate, cyclohexanecarboxylate and ptaquiloside.

A study was carried out to determine the cytotoxic effects of quercetin, shikimate, cyclohexane-carboxylate and ptaquiloside on Chinese hamster ovary (CHO), mouse fibroblast (3T3) and normal rat kidney (NRK) cells by cell growth inhibition assay of monolayers grown in 96-well plates. Molar concentrations inhibiting cell growth by 50% (IC50) after 48 h of incubation for quercetin, shikimate, cyclohexanecarboxylate and ptaquiloside were 0.8 x 10(-4)M, 0.8 x 10(-3)M, 0.4 x 10(-2) M and 0.6 x 10(-1)M respectively for CHO cells; 1.2 x 10(-4)M, 0.7 x 10(-3)M, 0.2 x 10(-7)M and 0.6 x 10(-1)M respectively for 3T3 cells; and 0.7 x 10(-4)M, 1.0 x 10(-3)M, 0.2 x 10(-2)M and 0.6 x 10(-1)M respectively for NRK cells. These inhibiting concentrations were extremely high when compared with mitomycin C whose cytotoxic effect occurred at 1 x 10(-6)M. Cyclohexanecarboxylate is a metabolite of shikimate; any possible cytotoxic effect of shikimate in vivo may be mediated by the major metabolite cyclohexanecarboxylate per se or after futher metabolism of this metabolite following absorption in animals. Quercetin, ptaquiloside and shikimate are constituents of bracken fern. The low order of cytotoxic activity of these constituents suggest that they are probably not directly the etiological agents of acute cattle bracken fern poisoning.

Animals↗
PubMed1988

Identification of the reactive cysteines of Escherichia coli 5-enolpyruvylshikimate-3-phosphate synthase and their nonessentiality for enzymatic catalysis.

Reaction of 5-enolpyruvylshikimate-3-phosphate synthase of Escherichia coli with the thiol reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) leads to a modification of only 2 of the 6 cysteines of the enzyme, with a significant loss of its enzymatic activity. Under denaturing conditions, however, all 6 cysteines of 5-enolpyruvylshikimate-3-phosphate synthase react with DTNB, indicating the absence of disulfide bridges in the native protein. In the presence of shikimate 3-phosphate and glyphosate, only 1 of the 2 cysteines reacts with the reagent, with no loss of activity, suggesting that only 1 of these cysteines is at or near the active site of the enzyme. Cyanolysis of the DTNB-inactivated enzyme with KCN leads to elimination of 5-thio-2-nitrobenzoate, with formation of the thiocyano-enzyme. The thiocyano-enzyme is fully active; it exhibits a small increase in its I50 for glyphosate (6-fold) and apparent Km for phosphoenolpyruvate (4-fold) compared to the unmodified enzyme. Its apparent Km for shikimate 3-phosphate is, however, unaltered. These results clearly establish the nonessentiality of the active site-reactive cysteine of E. coli 5-enolpyruvylshikimate-3-phosphate synthase for either catalysis or substrate binding. Perturbations in the kinetic constants for phosphoenolpyruvate and glyphosate suggest that the cysteine thiol is proximal to the binding site for these ligands. By N-[14C]ethylmaleimide labeling, tryptic mapping, and N-terminal sequencing, the 2 reactive cysteines have been identified as Cys408 and Cys288. The cysteine residue protected by glyphosate and shikimate 3-phosphate from its reaction with DTNB was found to be Cys408.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2004

Annotating the Plasmodium genome and the enigma of the shikimate pathway.

The completion of the Plasmodium falciparum genome sequence heralds a new era in the effort to identify all the parasite's genes along with their cellular functions. A combination of bioinformatics and experimental proof will facilitate this process. Many enzymes in metabolic processes have been identified, but several examples exist of incomplete pathways, such as the shikimate pathway. This review uses the example of the shikimate pathway to examine the application of bioinformatics to lead experimental design in post-genomic biology.

Amino Acid Sequence↗
PubMed1990

Active site labeling of the shikimate pathway enzyme, dehydroquinase. Evidence for a common substrate binding site within dehydroquinase and dehydroquinate synthase.

Dehydroquinase, the third enzyme of the shikimate biosynthetic pathway, is inactivated by iodoacetate. Iodoacetate behaves as an affinity label for the Escherichia coli enzyme with a Ki of 30 mM and a limiting inactivation rate of 0.014 min-1 at pH 7.0 and 25 degrees C. Affinity labeling is mediated by the negative charge of the reagent since iodoacetamide does not inactivate the enzyme. 2.1-2.3 mol of carboxymethyl groups are incorporated per mol of protein monomer resulting in 90% inactivation of enzymic activity. The majority of the bound label (80%) is split equally between 2 methionine residues, Met-23 and Met-205, which were identified by sequencing radiolabelled peptide fragments isolated after proteolytic digestion. An equilibrium mixture of the substrate (dehydroquinate) and product (dehydroshikimate) substantially reduces the inactivation rate and specifically decreases the incorporation of label at both of these site, implicating them as being in or near the active site of the enzyme. Sequence alignments with other biosynthetic dehydroquinases show that of the 2 methionine residues only Met-205 is conserved. N-terminal alignments of all the available dehydroquinase sequences (both catabolic and biosynthetic classes) revealed that Met-23, although itself not conserved, resides within a cluster of conserved sequence which may constitute part of the dehydroquinate binding site. A consensus sequence was derived from these alignments and used to probe the protein sequence data banks. A related sequence was found in dehydroquinate synthase, the enzyme which precedes dehydroquinase in the shikimate pathway. These results suggest that we have identified part of the dehydroquinate binding site in both enzymes.

Affinity Labels↗
PubMed2005

Evolution of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase-encoding genes in the yeast Saccharomyces cerevisiae.

The shikimate pathway resulting in three aromatic amino acids is initiated in different organisms by two and three 3-deoxy-d-arabino-heptulosonate-7-phosphate synthases, respectively. Aro3p and Aro4p are the yeast enzymes feedback-inhibited by phenylalanine and tyrosine, respectively. A yeast strain deficient in the general control transcriptional regulatory system of amino acid biosynthesis is unable to live in the presence of high amounts of phenylalanine and tyrosine. Here, we show that this yeast strain can be rescued by the expression of aroH from Escherichia coli encoding the tryptophan-regulated AroH as third isoenzyme. Yeast carrying Ec AroH as the only enzyme for the initial step of the shikimate pathway can grow in the absence of tryptophan. Without aromatic amino acids, this yeast strain survives only when the yeast ARO3 promoter instead of the ARO4 promoter drives E. coli aroH. The detailed analysis of Aro3p and Aro4p revealed a triple feedback control by tyrosine/phenylalanine and tryptophan. Dissecting this control allowed engineering of Aro4p S195A as an enzyme, which is inhibited like AroH only by tryptophan. In addition, Aro4p variants were constructed that show an equally strong inhibition by tyrosine and tryptophan (Aro4p P165G Q302R) and in which the regulation by tyrosine and tryptophan was reversed (Aro4p P165G). Our data suggest that yeast possesses only two instead of three isogenes encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate synthases because both isoenzymes can be fine tuned by tryptophan as additional effector and because transcriptional regulation by the general control system can be induced as backup when aromatic amino acids in the environment are imbalanced.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed2004

Aminomethylphosphonic acid, a metabolite of glyphosate, causes injury in glyphosate-treated, glyphosate-resistant soybean.

Glyphosate-resistant (GR) soybean [Glycine max (L.) Merr.] was developed by stable integration of a foreign gene that codes insensitive enzyme 5-enolpyruvylshikimate-3-phosphate synthase, an enzyme in the shikimate pathway, the target pathway of glyphosate. Application of glyphosate to GR soybean results in injury under certain conditions. It was hypothesized that if GR soybean is completely resistant to the glyphosate, injury could be caused by a metabolite of glyphosate, aminomethylphosphonic acid (AMPA), a known phytotoxin. Glyphosate and AMPA effects on one- to two-trifoliolate leaf stage (16-18-days old) GR and non-GR soybean were examined in the greenhouse. In GR soybean, a single application of glyphosate-isopropylammonium (1.12-13.44 kg/ha) with 0.5% Tween 20 did not significantly reduce the chlorophyll content of the second trifoliolate leaf at 7 days after treatment (DAT) or the shoot dry weight at 14 DAT compared with Tween 20 alone. A single application of AMPA (0.12-8.0 kg/ha) with 0.5% Tween 20 reduced the chlorophyll content of the second trifoliolate leaf by 0-52% at 4 DAT and reduced shoot fresh weight by 0-42% at 14 DAT in both GR and non-GR soybeans compared with Tween 20 alone. AMPA at 0.12 and 0.50 kg/ha produced injury in GR and non-GR soybean, respectively, similar to that caused by glyphosate-isopropylammonium at 13.44 kg/ha in GR soybean. AMPA levels found in AMPA-treated soybean of both types and in glyphosate-treated GR soybean correlated similarly with phytotoxicity. These results suggest that soybean injury to GR soybean from glyphosate is due to AMPA formed from glyphosate degradation.

Dose-Response Relationship, Drug↗
PubMedDate not supplied

The shikimate pathway. V. Fluorine-containing analogues of 3-deoxy-D-arabino hept-2-ulosonate-7-phosphate (DAHP).

(3R) and (3S) 3-deoxy-3-fluoro-7-phospho-D-arabino hept-2-ulosonic acids (3R and 3S-3F-DAHP) the 3-fluoro analogues of DAHP were synthesized from the corresponding 2-deoxy-2-fluoro hexose-6-phosphates. 3R- and 3S-3F-DAHP were tested as substrates for 3-dehydroquinate synthetase from E. coli. Determination of kinetic parameters showed that their apparent Km and Vm were in the same order of magnitude for these two compounds. Further conversion of 3R- and 3S-3F-DAHP into (6R) and (6S) 6-fluoro dehydroshikimate and (6R) and (6S) 6-fluoro shikimate, respectively, was investigated and results are discussed.

Chemical Phenomena↗
PubMed2000

Expression of tryptophan decarboxylase and tyrosine decarboxylase genes in tobacco results in altered biochemical and physiological phenotypes.

The substrate specificity of tryptophan (Trp) decarboxylase (TDC) for Trp and tyrosine (Tyr) decarboxylase (TYDC) for Tyr was used to modify the in vivo pools of these amino acids in transgenic tobacco. Expression of TDC and TYDC was shown to deplete the levels of Trp and Tyr, respectively, during seedling development. The creation of artificial metabolic sinks for Trp and Tyr also drastically affected the levels of phenylalanine, as well as those of the non-aromatic amino acids methionine, valine, and leucine. Transgenic seedlings also displayed a root-curling phenotype that directly correlated with the depletion of the Trp pool. Non-transformed control seedlings could be induced to display this phenotype after treatment with inhibitors of auxin translocation such as 2,3,5-triiodobenzoic acid or N-1-naphthylphthalamic acid. The depletion of aromatic amino acids was also correlated with increases in the activities of the shikimate and phenylpropanoid pathways in older, light-treated transgenic seedlings expressing TDC, TYDC, or both. These results provide in vivo confirmation that aromatic amino acids exert regulatory feedback control over carbon flux through the shikimate pathway, as well as affecting pathways outside of aromatic amino acid biosynthesis.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1980

The Site of the Inhibition of the Shikimate Pathway by Glyphosate: I. INHIBITION BY GLYPHOSATE OF PHENYLPROPANOID SYNTHESIS IN BUCKWHEAT (FAGOPYRUM ESCULENTUM MOENCH) .

The nonselective herbicide glyphosate (n-[phosphonomethyl]glycine) inhibited the light-induced accumulation of phenylpropanoid substances (chlorogenic acid, procyanidin, rutin, anthocyanin) in etiolated buckwheat hypocotyls 90% at 1 millimolar. Structurally related compounds, such as n,n-bis[phosphonomethyl]glycine, aminomethylphosphonate, methylglycine, and iminodiacetate, had little or no inhibiting effects. Of all amino acids tested, only l-phenylalanine reversed the inhibition, and partial reversal of anthocyanin synthesis was achieved with chorismate, phenylpyruvate, trans-cinnamate, p-coumarate, and naringenin. Phenylalanine concentrations were reduced in glyphosate-treated hypocotyls, and glyphosate effectively reduced the high level of phenylalanine that was caused by the phenylalanine ammonia-lyase inhibitor l-alpha-aminooxy-beta-phenylpropionate. Glyphosate had no significant effect on the time course of phenylalanine ammonia-lyase activity in hypocotyls incubated either in the dark or in the light. Under appropriate feeding conditions, glyphosate inhibited the incorporation of [(14)C]shikimate into all three aromatic amino acids, and radioactive shikimate accumulated in the tissue. The results lead to the conclusion that glyphosate interferes with the shikimate pathway at or prior to the formation of chorismate.

Journal Article↗
PubMed2004

Crystal structure of chorismate synthase: a novel FMN-binding protein fold and functional insights.

Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate to chorismate in the shikimate pathway, which represents an attractive target for discovering antimicrobial agents and herbicides. Chorismate serves as a common precursor for the synthesis of aromatic amino acids and many aromatic compounds in microorganisms and plants. Chorismate synthase requires reduced FMN as a cofactor but the catalyzed reaction involves no net redox change. Here, we have determined the crystal structure of chorismate synthase from Helicobacter pylori in both FMN-bound and FMN-free forms. It is a tetrameric enzyme, with each monomer possessing a novel "beta-alpha-beta sandwich fold". Highly conserved regions, including several flexible loops, cluster together around the bound FMN to form the active site. The unique FMN-binding site is formed largely by a single subunit, with a small contribution from a neighboring subunit. The isoalloxazine ring of the bound FMN is significantly non-planar. Our structure illuminates the essential functional roles played by the cofactor.

Amino Acid Sequence↗
PubMed2003

The crystal structure of shikimate dehydrogenase (AroE) reveals a unique NADPH binding mode.

Shikimate dehydrogenase catalyzes the NADPH-dependent reversible reduction of 3-dehydroshikimate to shikimate. We report the first X-ray structure of shikimate dehydrogenase from Haemophilus influenzae to 2.4-A resolution and its complex with NADPH to 1.95-A resolution. The molecule contains two domains, a catalytic domain with a novel open twisted alpha/beta motif and an NADPH binding domain with a typical Rossmann fold. The enzyme contains a unique glycine-rich P-loop with a conserved sequence motif, GAGGXX, that results in NADPH adopting a nonstandard binding mode with the nicotinamide and ribose moieties disordered in the binary complex. A deep pocket with a narrow entrance between the two domains, containing strictly conserved residues primarily contributed by the catalytic domain, is identified as a potential 3-dehydroshikimate binding pocket. The flexibility of the nicotinamide mononucleotide portion of NADPH may be necessary for the substrate 3-dehydroshikimate to enter the pocket and for the release of the product shikimate.

Alcohol Oxidoreductases↗
PubMed2001

The gene cluster for chloramphenicol biosynthesis in Streptomyces venezuelae ISP5230 includes novel shikimate pathway homologues and a monomodular non-ribosomal peptide synthetase gene.

Regions of the Streptomyces venezuelae ISP5230 chromosome flanking pabAB, an amino-deoxychorismate synthase gene needed for chloramphenicol (Cm) production, were examined for involvement in biosynthesis of the antibiotic. Three of four ORFs in the sequence downstream of pabAB resembled genes involved in the shikimate pathway. BLASTX searches of GenBank showed that the deduced amino acid sequences of ORF3 and ORF4 were similar to proteins encoded by monofunctional genes for chorismate mutase and prephenate dehydrogenase, respectively, while the sequence of the ORF5 product resembled deoxy-arabino-heptulosonate-7-phosphate (DAHP) synthase, the enzyme that initiates the shikimate pathway. A relationship to Cm biosynthesis was indicated by sequence similarities between the ORF6 product and membrane proteins associated with Cm export. BLASTX searches of GenBank for matches with the translated sequence of ORF1 in chromosomal DNA immediately upstream of pabAB did not detect products relevant to Cm biosynthesis. However, the presence of Cm biosynthesis genes in a 7.5 kb segment of the chromosome beyond ORF1 was inferred when conjugal transfer of the DNA into a blocked S. venezuelae mutant restored Cm production. Deletions in the 7.5 kb segment of the wild-type chromosome eliminated Cm production, confirming the presence of Cm biosynthesis genes in this region. Sequencing and analysis located five ORFs, one of which (ORF8) was deduced from BLAST searches of GenBank, and from characteristic motifs detected in alignments of its deduced amino acid sequence, to be a monomodular nonribosomal peptide synthetase. GenBank searches did not identify ORF7, but matched the translated sequences of ORFs 9, 10 and 11 with short-chain ketoreductases, the ATP-binding cassettes of ABC transporters, and coenzyme A ligases, respectively. As has been shown for ORF2, disrupting ORF3, ORF7, ORF8 or ORF9 blocked Cm production.

Amino Acid Sequence↗
PubMed1997

Biosynthetic Studies of omega-Cycloheptyl Fatty Acids in Alicyclobacillus cycloheptanicus. Formation of Cycloheptanecarboxylic Acid from Phenylacetic Acid.

The formation of the structurally novel, mono-substituted cycloheptane ring in omega-cycloheptyl fatty acids in Alicyclobacillus cycloheptanicus (formerly Bacillus cycloheptanicus) has been examined. Feeding experiments with (13)C- and (2)H-labeled intermediates demonstrated that cycloheptanecarboxylic acid (3), probably as its CoA thioester, is the starter unit for omega-cycloheptyl fatty acid biosynthesis. Analysis of the resultant labeling pattern from a feeding experiment with [U-(13)C(6)]glucose suggested a shikimate pathway origin of 3 via aromatic amino acids. [1,2-(13)C(2)]Phenylacetic acid (6) was efficiently metabolized into the 3-derived moiety in a manner reminiscent of the seven-membered ring Pseudomonas metabolite thiotropocin. The fates of the aromatic and benzylic hydrogens of 6 were determined; these dictated various boundary conditions for the biosynthetic pathway from 6 to 3. Taken together with the results from feeding experiments with postulated cycloheptenylcarboxylate biosynthetic intermediates, the data lead us to propose a pathway which involves an oxidative ring-expansion of 6 to a hydroxynorcaradiene intermediate followed by a series of double bond reductions and dehydrations to the saturated 3.

Journal Article↗
PubMed2006

Isotopic criteria in the characterization of aromatic molecules. 1. Hydrogen affiliation in natural benzenoid/phenylpropanoid molecules.

The site-specific isotope ratios of several families of aromatic molecules are analyzed in terms of hydrogen affiliation and discriminating potential. Among the aromatic molecules produced by plants, many are biosynthesized by the shikimate pathway, but the terpenic pathway also forms some compounds with a benzenic ring. In compounds of the phenylpropanoid family, specific hydrogen connections are determined with cinnamic acid, a key intermediate in the formation of a large number of aromatic molecules. Then affiliations through the phenylalanine precursor, back to the parent d-erythrose 4-phosphate and phosphoenolpyruvate molecules and finally to glucose, are considered. Typical isotopic profiles of the benzenic ring in natural, as compared to non-natural, molecules are defined. The dispersion observed in the (D/H)i ratios of the lateral chains is illustrative of diverse mechanistic responses and the role of exchange phenomena. The isotopic patterns of aromatic molecules pertaining to the terpenic family are drastically different from those of the shikimate descendants, and they exhibit much less variability. They enable the stereochemical affiliation of individual hydrogen atoms to be traced back first to the parent atoms in the common intermediate, geranyl diphosphate, then to the glyceraldehyde 3-phosphate and pyruvate couple involved in the DOXP pathway, and ultimately to the glucose precursor. The results illustrate the aptitude of the site-specific isotope ratios not only to authenticate natural with respect to chemical molecules but also to characterize different metabolic pathways and to reveal differences associated with the nature of the plant precursor.

Benzene↗
PubMed2001

Biochemical and X-ray crystallographic studies on shikimate kinase: the important structural role of the P-loop lysine.

Shikimate kinase, despite low sequence identity, has been shown to be structurally a member of the nucleoside monophosphate (NMP) kinase family, which includes adenylate kinase. In this paper we have explored the roles of residues in the P-loop of shikimate kinase, which forms the binding site for nucleotides and is one of the most conserved structural features in proteins. In common with many members of the P-loop family, shikimate kinase contains a cysteine residue 2 amino acids upstream of the essential lysine residue; the side chains of these residues are shown to form an ion pair. The C13S mutant of shikimate kinase was found to be enzymatically active, whereas the K15M mutant was inactive. However, the latter mutant had both increased thermostability and affinity for ATP when compared to the wild-type enzyme. The structure of the K15M mutant protein has been determined at 1.8 A, and shows that the organization of the P-loop and flanking regions is heavily disturbed. This indicates that, besides its role in catalysis, the P-loop lysine also has an important structural role. The structure of the K15M mutant also reveals that the formation of an additional arginine/aspartate ion pair is the most likely reason for its increased thermostability. From studies of ligand binding it appears that, like adenylate kinase, shikimate kinase binds substrates randomly and in a synergistic fashion, indicating that the two enzymes have similar catalytic mechanisms.

2,2'-Dipyridyl↗
PubMed1981

The aromatic amino acid pathway branches at L-arogenate in Euglena gracilis.

The recently characterized amino acid L-arogenate (Zamir et al., J. Am. Chem. Soc. 102:4499-4504, 1980) may be a precursor of either L-phenylalanine or L-tyrosine in nature. Euglena gracilis is the first example of an organism that uses L-arogenate as the sole precursor of both L-tyrosine and L-phenylalanine, thereby creating a pathway in which L-arogenate rather than prephenate becomes the metabolic branch point. E. gracilis ATCC 12796 was cultured in the light under myxotrophic conditions and harvested in late exponential phase before extract preparation for enzymological assays. Arogenate dehydrogenase was dependent upon nicotinamide adenine dinucleotide phosphate for activity. L-Tyrosine inhibited activity effectively with kinetics that were competitive with respect to L-arogenate and noncompetitive with respect to nicotinamide adenine dinucleotide phosphate. The possible inhibition of arogenate dehydratase by L-phenylalanine has not yet been determined. Beyond the latter uncertainty, the overall regulation of aromatic biosynthesis was studied through the characterization of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and chorismate mutase. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase was subject to noncompetitive inhibition by L-tyrosine with respect to either of the two substrates. Chorismate mutase was feedback inhibited with equal effectiveness by either L-tyrosine or L-phenylalanine. L-Tryptophan activated activity of chorismate mutase, a pH-dependent effect in which increased activation was dramatic above pH 7.8 L-Arogenate did not affect activity of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase or of chorismate mutase. Four species of prephenate aminotransferase activity were separated after ion-exchange chromatography. One aminotransferase exhibited a narrow range of substrate specificity, recognizing only the combination of L-glutamate with prephenate, phenylpyruvate, or 4-hydroxyphenylpyruvate. Possible natural relationships between Euglena spp. and fungi previously considered in the literature are discussed in terms of data currently available to define enzymological variation in the shikimate pathway.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1991

Stabilization of the shikimate pathway enzyme dehydroquinase by covalently bound ligand.

Reversible binding of a ligand to an enzyme active site can elicit a variety of changes in the protein, such as conformational changes (close to the site of binding or communicated over long distances), changes in the ionization state of surrounding amino acid side chains, changes in the interaction of the target protein with other subunits (or other proteins), or even changes in the thermodynamic stability of the protein. Relatively little attention has been given to studying these effects in proteins to which the ligand has been irreversibly bound, yet this can be a convenient way of studying the effects of ligand binding in the absence of association/dissociation equilibria. We report the dramatic changes which occur to the shikimate pathway enzyme dehydroquinase when ligand is attached to its active site after borohydride reduction of the mechanistically important Schiff's base intermediates. The effects of this modification have been characterized by limited proteolysis, circular dichroism, guanidine hydrochloride denaturation, and differential scanning calorimetry. The conclusions from these studies are that although anchoring the ligand at the active site does not cause a gross change in conformation, it does increase markedly the conformational stability of the protein. This is conclusively established by three separate experiments: 1) the modified protein is completely resistant to proteases, whereas the unmodified protein is very susceptible to proteolysis; 2) the concentration of guanidine hydrochloride required to unfold the ligand-linked dehydroquinase is 3-4-fold greater than that of the unmodified protein; 3) the melting temperature (Tm) of the modified protein is 40 degrees C higher than that of the unmodified protein. These results are a very clear example of the thermodynamic link between ligand binding, conformational stability, and proteolytic susceptibility in vitro and will be a useful system for dissecting the contributions of individual protein-ligand interactions to these parameters.

Calorimetry, Differential Scanning↗