Cloning and complete nucleotide sequence of the Escherichia coli glutamine permease operon (glnHPQ)

Summary The glutamine permease operon encoding the high-affinity transport system of glutamine in Escherichia coli could be cloned in one of the mini F plasmids, but not in pBR322 or pACYC184, by selection for restoration of the Gln⁺ phenotype, the ability to utilize glutamine as a sole carbon source. We determined the nucleotide sequence of the glutamine permease operon, which contains the structural gene of the periplasmic glutamine-binding protein (glnH), an indispensable component of the permease activity. The N-terminal amino acid sequence and the overall amino acid composition of the purified glutamine-binding protein were in good agreement with those predicted from the nucleotide sequence, if the N-terminal 22 amino acid residues were discounted. The latter comprised two Lys residues (nos. 2 and 6) followed by 16 hydrophobic amino acid residues and was assumed to be a signal peptide for transport into the periplasmic space. There were two additional reading frames (glnP and glnQ) downstream of glnH sharing a common promoter. It was concluded that the glnP and glnQ proteins as well as the glnH protein are essential for glutamine permease activity.     

A recombinant glutamine-binding protein from Escherichia coli: effect of ligand-binding on protein conformational dynamics

We have investigated the effect of the binding of glutamine on the conformational dynamics of the recombinant glutamine binding protein (GlnBP) from Escherichia coli by steady-state and time-resolved fluorescence techniques. The structural stability of the protein was also studied by far-UV circular dichroism spectroscopy in the range of temperature between 25 and 80 degrees C. The results showed that the interaction of the protein with the ligand resulted in a marked change of the structural and conformational dynamics features of the protein. In particular, the fluorescence and circular dichroism data showed that the presence of glutamine resulted in a dramatic increase of the protein thermal stability of about 10 degrees C. In addition, the fluorescence time-resolved data pointed out that both in the absence and in the presence of glutamine the protein structure was highly rigid with small amplitude of segmental motion up to 65 degrees C and a low accessibility of the protein tryptophan residues to acrylamide. The obtained results on the structural properties of the recombinant glutamine-binding protein in the absence and in the presence of glutamine can contribute to a better understanding of the transport-related functions of the protein and structurally similar periplasmic transport proteins, as well as to the design and development of new biotechnological applications of this class of proteins.     

Involvement of histidine and tryptophan residues of glutamine binding protein in the interaction with membrane-bound components of the glutamine transport system of Escherichia coli

We treated the glutamine binding protein with diethyl pyrocarbonate (DEPC) and N-bromosuccinimide (NBS) to modify respectively the sole histidine and tryptophan residues and examined the effect of these modifications on the ability of the binding protein to bind glutamine as well as the ability to restore glutamine transport in membrane vesicles of Escherichia coli. Under the conditions used, both DEPC and NBS markedly inhibited the ability to restore glutamine transport in vesicles without any significant effect on glutamine binding. Moreover, saturating quantities of glutamine had no protective effect on the inactivation of the binding protein by DEPC or NBS. Fluorometric measurement and amino acid analysis indicate that the inactivation of the binding protein in restoring vesicle transport by NBS can be attributed to the oxidation of a single tryptophan residue. Similar analysis and the inability of hydroxylamine to reverse the effect of DEPC indicate that the effects of DEPC can probably be attributed to alterations of the sole histidine and/or one or more lysine residues of the binding protein. We conclude that the glutamine binding protein possesses at least two largely nonoverlapping functional domains, one responsible for glutamine binding and the other for the interaction with the other components of the glutamine transport system.     

Nitrogen control of Salmonella typhimurium: co-regulation of synthesis of glutamine synthetase and amino acid transport systems.

Nitrogen control in Salmonella typhimurium is not limited to glutamine synthetase but affects, in addition, transport systems for histidine, glutamine, lysine-arginine-ornithine, and glutamate-aspartate. Synthesis of both glutamine synthetase and transport proteins is elevated by limitation of nitrogen in the growth medium or as a result of nitrogen (N)-regulatory mutations. Increases in the amounts of these proteins were demonstrated by direct measurements of their activities, by immunological techniques, and by visual inspection of cell fractions after gel electrophoresis. The N-regulatory mutations are closely linked on the chromosome to the structural gene for glutamine synthetase, glnA: we discuss the possibility that they lie in a regulatory gene, glnR, which is distinct from glnA. Increases in amino acid transport in N-regulatory mutant strains were indicated by increased activity in direct transport assays, improved growth on substrates of the transport systems, and increased sensitivity to inhibitory analogs that are trnasported by these systems. Mutations to loss of function of individual transport components (hisJ, hisP, glnH, argT) were introduced into N-regulatory mutant strains to determine the roles of these components in the phenotype and transport behavior of the strains. The structural gene for the periplasmic glutamine-binding protein, glnH, was identified, as was a gene argT that probably encodes the structure of the lysine-arginine-ornithine-binding protein. Genes encoding the structures of the histidine- and glutamine-binding proteins are not linked to glnA or to each other by P22-mediated transduction; thus, nitrogen control is exerted on several unlinked genes.     

The differences in the microenvironment of the two tryptophan residues of the glutamine-binding protein from Escherichia coli shed light on the binding properties and the structural dynamics of the protein

Glutamine-binding protein (GlnBP) from Escherichia coli is a monomer (26 kDa) that is responsible for the first step in the active transport of L-glutamine across the cytoplasmic membrane. GlnBP consists of two domains (termed large and small) linked by two antiparallel beta-strands. The large domain is similar to the small domain but it contains two additional alpha-helices and three more short antiparallel beta-strands. The deep cleft formed between the two domains contains the ligand-binding site. The binding of L-glutamine leads to cleft closing and a significant structural change with the formation of the so-called "closed form" structure. The protein contains two tryptophan residues (W32 and W220) and 10 tyrosine residues. We used phosphorescence spectroscopy measurements to characterize the role of the two tryptophan residues in the protein structure in the absence and the presence of glutamine. Our results pointed out that the phosphorescence of GlnBP is easily detected in fluid solutions where the emission of the two tryptophan residues is readily discriminated by the drastic difference in the phosphorescence lifetime allowing the assignments of the short lifetime to W220 and the long lifetime to W32. In addition, our results showed that the triplet lifetime of the superficial W220 is unusually short because of intramolecular quenching by the proximal Y163. On the contrary, the lifetime of W32 is several hundred milliseconds long, implicating a well-ordered, compact fold of the surrounding polypeptide. The spectroscopic data were analyzed and discussed together with a detailed inspection of the 3D structure of GlnBP.     

Proton nuclear magnetic resonance studies on glutamine-binding protein from Escherichia coli. Formation of intermolecular and intramolecular hydrogen bonds upon ligand binding

Proton nuclear magnetic resonance studies have revealed several structural and dynamic properties of the glutamine-binding protein of Escherichia coli. When this protein binds L-glutamine, six low-field, exchangeable proton resonances appear in the region from +5.5 to +10 parts per million downfield from water (or +10.2 to +14.7 parts per million downfield from the methyl proton resonance of 2,2-dimethyl-2-silapentane-5-sulfonate). This suggests that the binding of L-glutamine induces specific conformational changes in the protein molecule, involving the formation of intermolecular and intramolecular hydrogen bonds between the glutamine-binding protein and L-glutamine, and within the protein molecule. The oxygen atom of the gamma-carbonyl group of L-glutamine is likely to be involved in the formation of an intermolecular hydrogen bond between the ligand and the binding protein. We have shown that at least one phenylalanine and one methyl-containing residue are spatially close to this intermolecular hydrogen-bonded proton. The intermolecular and intramolecular hydrogen-bonded protons of the ligand-protein complex undergo solvent exchange. The local conformations around these intermolecular and intramolecular hydrogen bonds are quite stable when subjected to pH and temperature variations. From these results, the utility of proton nuclear magnetic resonance spectroscopy for investigating such binding proteins has been shown, and a picture of the ligand-binding process can be drawn.     

States of tryptophan residues in castor bean hemagglutinin: the perturbing effects of solvents on tryptophans in hemagglutinin and its complexes as analyzed by UV-difference spectroscopy

The states of tryptophan residues in castor bean hemagglutinin (CBH) were analyzed by solvent perturbation studies employing ultraviolet difference spectroscopy. Eight out of 22 tryptophan residues in CBH were exposed to ethylene glycol and glycerol, suggesting that the remaining 14 tryptophan residues are buried in the interior of the CBH molecule. The fraction of tryptophan residues accessible to the perturbant decreased with increase in the molecular size of the perturbant, and only 2 tryptophan residues were exposed to polyethylene glycol 600. Upon binding with raffinose, 2 tryptophan residues were shielded from the perturbing effect of the solvent, and binding of lactose reduced the number of tryptophan residues accessible to the perturbant by 1 mol per mol of protein. Binding of galactose, however, did not change the accessibility of tryptophan to the perturbant. On the other hand, the accessibility of tyrosine to the perturbant remained unchanged after binding with raffinose and lactose, suggesting that tyrosine is not directly involved in the saccharide binding of CBH. Based on these results, it is proposed that one tryptophan residue at the saccharide-binding site on each B-chain of CBH lies on the surface of the protein molecule and is located at a subsite which is accessible to a glucopyranoside moiety in the lactose molecule or a glycopyranosyl-fructofuranosyl moiety in the raffinose molecule, whereas such a residue is not present at the galactopyranoside-recognition site.     

Experimental allergic encephalitis: study of cellular immunity to the encephalitogenic determinant.

Guinea pigs were tested for cellular immunity to the encephalitogenic tryptophan peptide, the major encephalitogenic determinant of central nervous system basic protein, representing residues 114 to 122 of the molecule. Guinea pigs sensitized with human basic protein regularly developed experimental allergic encephalitis, but did not show cellular immunity to the encephalitogenic tryptophan peptide as measured by skin test reactivity, lymphocyte stimulation, or macrophage migration inhibition, although they did show cellular immunity to the immunizing antigen, human basic protein. Animals sensitized with the synthetic tryptophan peptide also regularly develop clinical and histologic features of experimental allergic encephalitis, and show cellular immunity to the peptide but not to human basic protein. The work of others indicates that, in guinea pigs sensitized with the whole basic protein, there are determinants for cellular immunity located near the encephalitogenic tryptophan peptide. The test peptides used in these studies all included amino acid residues of the basic protein not included in the encephalitogenic tryptophan peptide used in our study. Our work indicates that the encephalitogenic peptide is not one of the determinants for cellular immunity in the basic protein molecule. Since cellular immunity to the disease-producing determinant of the molecule could not be demonstrated, this work further suggests that cellular immunity, as measured by the three tests described herein, may not necessarily be correlated with production of experimental allergic encephalitis.     

The structure and stability of the glutamine-binding protein from Escherichia coli and its complex with glutamine

A study was made of the conformational changes in the Escherichia coli glutamine-binding potein (GlnBP) induced by GdnHCl, and of the effect of glutamine (Gln) binding on these processes. Intrinsic fluorescence, ANS emission fluorescence, and far- and near-UV circular dichroism spectroscopy were used. The obtained experimental data were interpreted, taking into the account results of the analysis of tryptophan and tyrosine residues microenvironments. This enabled us to explain the negligible contribution of Tyr residues to the bulk fluorescence of the native protein, the similarity of fluorescence characteristics of GlnBP and GlnBP/Gln, and an uncommon effect of the excess of fluorescence intensity at 365 nm (Trp emission) upon excitation at 297 nm compared to the excitation at 280 nm. The latter effect is explained by the spectral dependence of Trp 32 and Trp 220 contributions to protein absorption. The dependence of Trp fluorescence of protein on the excitation wavelength must be taken into account for the evaluation of Tyr residues contribution to the bulk fluorescence of protein, and in principle, it may also be used for the development of an approach to decomposition of multi-component protein fluorescence spectrum. The parametric presentation of fluorescence data showed that both GlnBP unfolding and GlnBP/Gln unfolding are three-step processes (N-->I1-->I2-->U), though in the case of the GlnBP/Gln complex these stages essentially overlap. Despite its complex character, GlnBP unfolding is completely reversible. In comparison with GlnBP, in the case of GlnBP/Gln the dramatic shift of N-->I1 process to higher GdHCl concentrations is shown.     

Cation-pi interactions as determinants for binding of the compatible solutes glycine betaine and proline betaine by the periplasmic ligand-binding protein ProX from Escherichia coli

Compatible solutes such as glycine betaine and proline betaine are accumulated to exceedingly high intracellular levels by many organisms in response to high osmolarity to offset the loss of cell water. They are excluded from the immediate hydration shell of proteins and thereby stabilize their native structure. Despite their exclusion from protein surfaces, the periplasmic ligand-binding protein ProX from the Escherichia coli ATP-binding cassette transport system ProU binds the compatible solutes glycine betaine and proline betaine with high affinity and specificity. To understand the mechanism of compatible solute binding, we determined the high resolution structure of ProX in complex with its ligands glycine betaine and proline betaine. This crystallographic study revealed that cation-pi interactions between the positive charge of the quaternary amine of the ligands and three tryptophan residues forming a rectangular aromatic box are the key determinants of the high affinity binding of compatible solutes by ProX. The structural analysis was combined with site-directed mutagenesis of the ligand binding pocket to estimate the contributions of the tryptophan residues involved in binding.     

Unfolding and refolding of the glutamine-binding protein from Escherichia coli and its complex with glutamine induced by guanidine hydrochloride

The aim of this work was to study the conformational changes of the Escherichia coli glutamine-binding protein (GlnBP) induced by GdnHCl and the effect of the binding of glutamine (Gln) on these processes. To this end, GdnHCl-induced unfolding of GlnBP alone and its GlnBP-Gln complex was studied by protein intrinsic fluorescence, ANS emission fluorescence, and far- and near-UV circular dichroism spectroscopy. The obtained spectroscopic data were interpreted taking into the account the peculiarities of protein three-dimensional structure. In particular, the fact that formation of a complex of GlnBP and Gln, which essentially changes the global structure of protein, affects only insignificantly the microenvironments of tryptophan residues elucidates the similarity of the emission spectra of GlnBP and the GlnBP-Gln complex, and the existence of quenching groups near tyrosine residues and an effective nonradiative Tyr --> Trp and/or Tyr --> Tyr --> Trp energy transfer provide an explanation for the negligibly small contribution of tyrosine to the bulk fluorescence of the native protein and for its increase in protein unfolding. The use of the parametric presentation of fluorescence data showed that both GlnBP unfolding and GlnBP-Gln unfolding are three-step processes (N --> I(1) --> I(2) --> U), though in the case of the GlnBP-Gln complex these stages essentially overlap. Despite the complex character, GlnBP unfolding is completely reversible. The dramatic shift of the N --> I(1) process to higher GdnHCl concentrations for the GlnBP-Gln complex in comparison with GlnBP was shown.     

Yeast 5S rRNA binding to ribosomal protein L1a alters the fluorescence of tryptophan residues lying outside the binding site.

The yeast ribosomal protein L1a contains two tryptophan residues located at positions 95 and 183. Spectrofluorometric analysis showed that the average tryptophan environment is moderately polar. Quenching studies of the yeast 5S rRNA-L1a protein complex (RNP) with acrylamide and iodide revealed tryptophan heterogeneity. The two tryptophan residues are located in the non-RNA-binding region of the L1a molecule. However, dissociation of the yeast 5S rRNA-L1a protein RNP complex to its components resulted in a decline of tryptophan fluorescence. The observation implied that the environment of the tryptophan-containing L1a regions which were not known to be involved in RNA binding was influenced by association with the 5S rRNA molecule.     

Regulation of a common amidotransferase subunit.

In Bacillus subtilis the trpX locus specifies a glutamine-binding protein designated subunit X, which forms a complex with subunit E to constitute the anthranilate synthase enzyme aggregate (EX) and subunit A to constitute the p-aminobenzoate synthase enzyme aggregate (AX). Subunit X confers upon these enzyme complexes the ability to utilize glutamine as a substrate. The trpX locus has been examined to determine its map position and control. (i) The trpX locus was found to be cotransformed with the lysS and pabA loci. The results of three-factor transformation analyses suggest the following order of these markers: lysS-sul-trpX-pabA. (ii) Mutation to constitutivity of the tryptophan operon resulted in a 50- to 60-fold increase in the level of subunit X when the mutant contained functional trE and abA gene products; however, in the absence of subunit E there was only a 4- to 5-fold increase in the glutamine-binding protein. (iii) Formation of subunit X was derepressed under conditions that allow for the derepression of the trpE and/or pabA loci. (iv) Subunit X synthesis was derepressed to a greater extent in mutants that contain a functional trpE gene product than in mutants that contain a nonsense mutation in the trpE locus. These results are consistent with the hypothesis that the trpE and pabA gene products affect the expression and control of the trpX locus.     

Urea cycle disorders, hyperammonemia and neurotransmitter changes

In congenital urea cycle disorders, detoxification of ammonia is impaired, leading to hyperammonemia. Ammonia is the major component causing the acute neurological disturbances. It may influence the supply of substrate and its transport at the blood-brain barrier (BBB) which results in alterations in the synthesis and catabolism of neurotransmitters in the brain. In hyperammonemic rats, the uptake of tryptophan into the brain is increased with an augmented flux through the serotonin pathway. In the forebrain, glutamine as well as amino acids transported with the same L-carrier system, such as phenylalanine, tyrosine and tryptophan, are elevated. It is postulated that the increased transport of tryptophan at the BBB occurs in exchange with glutamine. Methionine sulfoximine (MSO) inhibits glutamine synthetase in the cerebral cortex. The activity drops from 5.85 +/- 0.38 to 1.07 +/- 0.37 mumol/min/g wet weight. Under MSO, the brain tryptophan uptake also decreased to 64.2 +/- 4.5% in hyperammonemic rats, to 54.1 +/- 8.0% in untreated hyperammonemic rats, whereas without MSO an increase of tryptophan uptake was observed. An effect of glutamine on tryptophan transport could also be demonstrated using brain microvessel preparations as a model for the BBB. Our findings indicate that preloading isolated microvessels with L-glutamine increases tryptophan uptake into the endothelia when L-glutamine is at concentrations found in brain homogenates under hyperammonemia. Since brain microvessels do not contain glutamine synthetase activity, enzymes from the gamma-glutamyl cycle may be involved in the glutamine-mediated tryptophan transport.(ABSTRACT TRUNCATED AT 250 WORDS)     

Model structures of the N-methyl-D-aspartate receptor subunit NR1 explain the molecular recognition of agonist and antagonist ligands

Molecular models of the ligand-binding domain of N-methyl-d-aspartate subunit R1 (NR1) were made using the published crystal structures of rat glutamate receptor B (GluRB), the bacterial glutamate receptor (GluR0), and the glutamine-binding protein (QBP) of Escherichia coli. Separate models of NR1 were built to represent the ligand-binding conformation for agonist (glycine, d- and l-isomers of serine and alanine, and the partial agonist ligand d-cycloserine) and antagonist (5,7-dichloro-4-oxo-1,4-dihydroquinoline-2-carboxylic acid (DCKA) and E-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1-H-indole-2-carboxylic acid (MDL 105,519)) ligands. Side-chain conformations of residues within the NR1 ligand-binding site were selected that optimized the hydrophobic packing and hydrogen bonding among residues, while taking into account published data comparing receptor mutants with wild-type NR1. Ligands docked to the model structures provide a rational explanation for the observed differences in binding affinity and receptor activation among agonist and antagonist ligands. NR1 prefers smaller ligands (glycine, serine, and alanine) in comparison with GluRB and GluR0 that bind l-glutamate: the bulky side chain of W731 in NR1 dramatically reduces the size of the ligand-binding site, functioning to selectively restrict recognition to glycine and the d-isomers of serine and alanine. Nevertheless, many of the interactions seen for ligands bound to GluRB, GluR0, and periplasmic-binding proteins are present for the ligands docked to the model structures of NR1.     

Crystal structure of the human receptor activity-modifying protein 1 extracellular domain

Receptor activity-modifying protein (RAMP) 1 forms a heterodimer with calcitonin receptor-like receptor (CRLR) and regulates its transport to the cell surface. The CRLR.RAMP1 heterodimer functions as a specific receptor for calcitonin gene-related peptide (CGRP). Here, we report the crystal structure of the human RAMP1 extracellular domain. The RAMP1 structure is a three-helix bundle that is stabilized by three disulfide bonds. The RAMP1 residues important for cell-surface expression of the CRLR.RAMP1 heterodimer are clustered to form a hydrophobic patch on the molecular surface. The hydrophobic patch is located near the tryptophan residue essential for binding of the CGRP antagonist, BIBN4096BS. These results suggest that the hydrophobic patch participates in the interaction with CRLR and the formation of the ligand-binding pocket when it forms the CRLR.RAMP1 heterodimer.     

Mutation analysis of carbamoyl phosphate synthetase: Does the structurally conserved glutamine amidotransferase triad act as a functional dyad?

Evolutionarily conserved triad glutamine amidotransferase (GAT) domains catalyze the cleavage of glutamine to yield ammonia and sequester the ammonia in a tunnel until delivery to a variety of acceptor substrates in synthetase domains of variable structure. Whereas a conserved hydrolytic triad (Cys/His/Glu) is observed in the solved GAT structures, the specificity pocket for glutamine is not apparent, presumably because its formation is dependent on the conformational change that couples acceptor availability to a greatly increased rate of glutamine cleavage. In Escherichia coli carbamoyl phosphate synthetase (eCPS), one of the best characterized triad GAT members, the Cys269 and His353 triad residues are essential for glutamine hydrolysis, whereas Glu355 is not critical for eCPS activity. To further define the glutamine-binding pocket and possibly identify an alternative member of the catalytic triad that is situated for this role in the coupled conformation, we have analyzed mutations at Gln310, Asn311, Asp334, and Gln351, four conserved, but not yet analyzed residues that might potentially function as the third triad member. Alanine substitution of Gln351, Asn311, and Gln310 yielded respective K(m) increases of 145, 27, and 15, suggesting that Gln351 plays a key role in glutamine binding in the coupled conformation, and that Asn311 and Gln310 make less significant contributions. None of the mutant k (cat) values varied significantly from those for wild-type eCPS. Combined with previously reported data on other conserved eCPS residues, these results strongly suggest that Cys269 and His353 function as a catalytic dyad in the GAT site of eCPS.     

Atomic interactions in protein-carbohydrate complexes. Tryptophan residues in the periplasmic maltodextrin receptor for active transport and chemotaxis.

We have refined the 1.9 A resolution crystal structures of two maltodextrin receptor mutants in which tryptophan residues 230 and 232 have been changed to alanine and compared these structures with the refined 1.7 A structure of the wild-type protein. In the wild-type structure, Trp230, which is located in the maltodextrin-binding groove, stacks against the B-face of the reducing sugar of the bound maltose. Trp232, which is located near the protein surface, does not participate directly in sugar binding. Relative to the wild-type structure, neither mutation caused a significant rearrangement in the overall protein structure or in the mode of binding maltose. Although the position once occupied by Trp230 remains empty, a new water molecule has moved near the void. In contrast, a new water molecule has entered into the space once occupied by Trp232. Whereas one hydrogen bond is formed with the water molecule near the Trp230 void, no hydrogen bond is associated with the water molecule occupying the space vacated by Trp232. The three van der Waals' contacts between Trp230 and maltose in the wild-type structure that are lost in the W230A mutation could contribute to the 12-fold decrease in ligand-binding activity of the mutant protein. The W232A mutation causes little change in binding activity. The structures of these mutant proteins also provide some insight into the complicated tryptophan fluorescence spectra of the maltodextrin binding-protein. The change in fluorescence due to the deletion of Trp230 can readily be explained as resulting directly from loss of Trp230 in the sugar-binding site. The change in fluorescence due to deletion of Trp232, however, is ascribed to the modification of local interactions mediated by the binding of maltodextrin since the tryptophan is not directly involved in any sugar-binding interaction.     

The glutamine residues reactive in transglutaminase-catalyzed cross-linking of involucrin

The protein involucrin, synthesized by human keratinocytes, contains 585 amino acids, largely in the form of 10 amino acid repeats, each containing glutamines in 3 conserved positions. Involucrin is a substrate for the keratinocyte transglutaminase and is labeled by the cosubstrate amine, glycine ethyl ester. Study of tryptic peptides of involucrin shows that a single glutamine (residue 496), located 89 residues from the C-terminal end, is preferentially labeled by the enzyme. Additional glutamine residues become reactive when the molecule is fragmented. The C-terminal end, isolated as a cyanogen bromide fragment of 275 residues, is labeled equally at 2 glutamine residues. The polypeptide containing residues 148 to 280 accepts practically no amine while in intact involucrin but as a free fragment is labeled at multiple glutamine residues. It is concluded that the C-terminal and N-terminal ends of the protein are directive influences in that they suppress the reactivity of a number of glutamine residues in the intact molecule, leaving one glutamine highly preferred by the transglutaminase.     

Chemical modification studies on Abrus agglutinin. Involvement of tryptophan residues in sugar binding.

The galactose-binding lectin from the seeds of the jequirity plant (Abrus precatorius) was subjected to various chemical modifications in order to detect the amino acid residues involved in its binding activity. Modification of lysine, tyrosine, arginine, histidine, glutamic acid and aspartic acid residues did not affect the carbohydrate-binding activity of the agglutinin. However, modification of tryptophan residues carried out in native and denaturing conditions with N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide led to a complete loss of its carbohydrate-binding activity. Under denaturing conditions 30 tryptophan residues/molecule were modified by both reagents, whereas only 16 and 18 residues/molecule were available for modification by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide respectively under native conditions. The relative loss in haemagglutinating activity after the modification of tryptophan residues indicates that two residues/molecule are required for the carbohydrate-binding activity of the agglutinin. A partial protection was observed in the presence of saturating concentrations of lactose (0.15 M). The decrease in fluorescence intensity of Abrus agglutinin on modification of tryptophan residues is linear in the absence of lactose and shows a biphasic pattern in the presence of lactose, indicating that tryptophan residues go from a similar to a different molecular environment on saccharide binding. The secondary structure of the protein remains practically unchanged upon modification of tryptophan residues, as indicated by c.d. and immunodiffusion studies, confirming that the loss in activity is due to modification only.