The amino acid sequence of glutamate decarboxylase from Escherichia coli. Evolutionary relationship between mammalian and bacterial enzymes.

The amino acid sequence of glutamate decarboxylase from Escherichia coli was solved by a combination of automated Edman degradation of peptide fragments derived by proteolytic and chemical cleavage and sequencing of DNA. Correct alignment of three peptides, for which no peptide overlaps were available, was achieved by sequencing a 1.1-kbp fragment of DNA produced by a polymerase-chain reaction using primers corresponding to sequences known to be in amino-terminal and carboxy-terminal regions of the protein. Sequence similarity (24% identity) with mammalian glutamate decarboxylase was found to be limited to a 55-residue sequence around the lysine residue that binds the coenzyme. Stronger similarity (38% identity), again confined to the same region, is seen with bacterial pyridoxal-phosphate-dependent histidine decarboxylase.     

Purification of plasma membrane penicillinase from Bacillus licheniformis 749/C and comparison with exoenzyme.

The membrane penicillinase of Bacillus licheniformis 749/C has been demonstrated to be a phospholipoprotein. The homogeneous enzyme gives a positive reaction for phosphorous and for unsaturated fatty acids, has a molecular weight of 33,000 in contrast to 29,000 for the exoenzyme, and contains 8 to 9 additional residues of aspartate or asparagine, 4 to 5 of serine, 7 of glutamate or glutamine, and 4 to 5 of glycine per mole. The COOH-terminal sequence of both membrane and exoenzymes is -Met-Asn-Gln-Lys-COOH; hence the extra peptide portion present in the membrane enzyme is not attached to the COOH-terminus of the exoenzyme. Procedures which readily detected the lysine residue at the NH2 terminus of the exoenzyme did not yield a positive test with the membrane form. The NH2 terminus of the membrane enzyme may be blocked by or linked to the phospholipid. A procedure for the preparation of membrane penicillinase on a large scale and an improved method for purification of the exoenzyme have been developed.     

Substrate specificity of the oncoprotein v-Fps: site-specific mutagenesis of the putative P+1 pocket

Based on the X-ray structure of the insulin receptor kinase [Hubbard, S. R. (1997) EMBO J. 16, 5572-5581], Arg-1130 in the oncoprotein v-Fps, a nonreceptor tyrosine protein kinase, is predicted to interact with the P+1 glutamate in substrate peptides. To determine whether this residue is an important recognition element in v-Fps, Arg-1130 was substituted with leucine (R1130L) and glutamic acid (R1130E). The ability of these mutants to phosphorylate the peptide EAEIYXAIE, where X is glutamic acid, alanine, or lysine, was assessed. A comparison of the rates of peptide phosphorylation under limiting substrate concentrations (i.e., k(cat)/K(m) conditions) indicates that substrate specificity is altered by the electrostatic environment of the P+1 pocket. When the pocket displays a positive charge (Arg-1130; wild type), no charge (R1130L), or a negative charge (R1130E), v-Fps prefers to phosphorylate the glutamate peptide over the lysine peptide by a 200:1, 9:1, or 1:1 margin. While k(cat)/K(m) for the glutamate peptide is 50-fold higher for wild type compared to R1130E, k(cat)/K(m) for the lysine peptide is 3-fold higher for R1130E compared to wild type, a 150-fold change in relative substrate specificity. Analysis of the individual steps in the kinetic mechanism using viscosometric techniques indicates that the wild-type enzyme binds the glutamate peptide 3-fold better than the alanine peptide and, at least, 10-fold better than the lysine peptide. For R1130L, this margin range is reduced substantially, and for R1130E, no binding preference is observed. Nonetheless, the lysine peptide binds, at least, 4-fold better to R1130E than to wild type, and the glutamate peptide binds 3-fold poorer to R1130E than to wild type. The mutants lower the phosphoryl transfer rate by 4-30-fold for the three peptides, suggesting that Arg-1130 helps to position the tyrosine for optimum catalysis. The data indicate that a single mutation in v-Fps can alter significantly the relative substrate specificity by about 2 orders of magnitude with, at least, 50% of this effect occurring through relative changes in peptide binding affinity.     

Analysis of the importance of arginine 102 in neutral endopeptidase (enkephalinase) catalysis.

Neutral endopeptidase 24.11 contains an active site arginine believed to function in substrate binding. This arginine is thought to form an ionic interaction with the COOH-terminal carboxylate of NEP substrates. The functionality of arginine 102 has been investigated by using site-directed mutagenesis to produce mutants in which this residue was converted to a lysine, glycine, glutamine, or glutamate. All of the mutants exhibited essentially full activity as determined with a synthetic peptide amide, glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide. In contrast, activity was detected only with the wild-type enzyme and the lysine mutant using a synthetic substrate containing a free COOH-terminal carboxylate, dansyl-Gly-Trp-Gly. Inhibition studies with the physiologically active peptide substrates substance P, endothelin, and angiotensin I, as well as substance P free acid, [D-Ala2,Leu5]enkephalin, and [D-Ala2,Leu5]enkephalinamide indicated a lack of importance of arginine 102 in substrate binding. With [D-Ala2,Met5]enkephalin and the chemotactic peptide, N-formyl-Met-Leu-Phe, a significant decrease in affinity is observed with the arginine 102 mutants. These results suggest that the contribution of arginine 102 to substrate binding is dependent upon the strength of other subsite interactions. Examination of dipeptides as inhibitors indicates that the nature and orientation of the P'2 residue is important in determining the strength of the interaction of arginine 102 with its substrates.     

The Positional Specificity of EXXK Motifs within an Amphipathic α-Helix Dictates Preferential Lysine Modification by Acrolein: Implications for the Design of High-Density Lipoprotein Mimetic Peptides

Despite the ability of acrolein to damage proteins, factors governing its reactivity with the ε-amino group of lysine are poorly understood. We used a small 26-mer α-helical peptide (ATI-5261) to evaluate the influence of acidic glutamate (E) residues on site-specific lysine modification by acrolein and if this targeting played a major role in inhibiting the cholesterol efflux activity of the peptide. Exposure of ATI-5261 to acrolein resulted in N-(3-formyl-3,4-dehydropiperidino) (FDP)-lysine adducts at positions 5 and 25 and led to a concentration-dependent reduction in cholesterol efflux activity (55 ± 7 and 83 ± 3% decrease with 5:1 and 20:1 acrolein:peptide molar ratios, respectively). Amino acid substitution (K → R) experiments and mass spectrometry revealed neither K5 nor K25 was preferentially modified by acrolein, despite the location of K5 within a putative EXXK motif. Moreover, both lysine residues remained equally reactive when the lipidated peptide was exposed to acrolein. In contrast, placement of EXXK in the center of ATI-5261 resulted in site-specific modification of lysine. The latter was dependent on glutamate, thus establishing that acidic residues facilitate lysine modification and form the molecular basis of the EXXK motif. Preferential targeting of lysine, however, failed to augment the inhibitory effect of the aldehyde. Overall, the inhibitory effects of acrolein on cholesterol efflux activity were largely dependent on the number of lysine residue modifications and cross-linking of α-helical strands that restricted dissociation of the peptide to active forms.     

Enzymatic synthesis of vasoactive intestinal peptide analogs by transglutaminase.

Vasoactive intestinal peptide is an amino acceptor and donor substrate for tissue transglutaminase (TGase) in vitro. This peptide contains a single glutamine residue, Gln16, which was identified as the amino acceptor substrate. Different gamma(glutamyl16)amine derivatives of vasoactive intestinal peptide were synthesized enzymatically in vitro. The modification is very fast when compared with that of many native substrates of TGase. The analogs 1,3-diaminopropane, putrescine, cadaverine, spermidine, spermine, glycine ethyl ester and mono-dansylcadaverine of the peptide were purified by high-performance liquid chromatography on a reverse-phase column and were analyzed by electrospray mass spectrometry. When amines were absent in the assay mixture as an external amino donor, lysine residue occurring in the peptide was an effective amino donor site for TGase. Only one of the three lysine residues of vasoactive intestinal peptide, namely Lys21, was demonstrated to be involved in both inter- and intramolecular cross-link formation.     

The reactivity of functional groups as a probe for investigating the topography of tobacco mosaic virus. The use of mutants with additional lysine residues in the coat protein

Several mutants of tobacco mosaic virus that contain additional lysine residues as a result of mutations in the coat protein were investigated. Mutant E66 has a lysine residue replacing asparagine at position 140 when compared with the wild-type vulgare and this lysine residue reacts readily in the intact virus with methyl picolinimidate. Mutant B13a has two new lysine residues in the coat protein, replacing a glutamine at position 9 and an asparagine at position 33, whereas mutant B13b has the single replacement of glutamine by lysine at position 9. The lysine residue at position 9 in mutants B13a and B13b also reacts readily with methyl picolinimidate in the intact virus but the lysine at position 33 in mutant B13a did not react under these conditions. However, when the isolated coat protein from mutant B13a was treated with methyl picolinimidate, the lysine residue at position 33 did become modified, showing that the loss in reactivity of this residue towards the imidoester in the intact virus is a result of the assembly of the protein subunit into the virus structure. These results are compatible with and extend previous studies on the sero-logical properties of mutants of tobacco mosaic virus and illustrate the value of methyl picolinimidate as a reagent for probing the accessibility of amino groups in proteins. When intact tobacco mosaic virus (vulgare) was treated with p-iodobenzenesulphonyl chloride, no reaction with the lysine residues at positions 33 or 68 in the virus subunit could be detected but complete modification of tyrosine-139 was achieved. This result also extends previous studies with other reagents. The usefulness of the differential reactivity of the lysine residues in tobacco mosaic virus and its mutants as a means of attaching heavy-atom labels at chemically defined positions for subsequent X-ray-diffraction analysis and the implications of these experiments for deciphering the folding of the peptide chain in the virus subunit are discussed.     

Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora. IV. The COOH-terminal 669 residues of the peptide chain; comparison with other glutamate dehydrogenases.

A sequence is presented for the COOH-terminal 669 residues of the NAD-specific glutamate dehydrogenase of Neurospora crassa. Comparison of this sequence with those of the vertebrate glutamate dehydrogenases of chicken and bovine liver and with the NADP-specific enzyme of Neurospora shows some similarities in sequences around residues previously identified as important for the function of these enzymes. These are: (a) the reactive lysine residue of low pK in the NADP and the vertebrate enzymes; (b) the tyrosine residue of the NADP enzyme that is readily nitrated by tetranitromethane with inactivation, a residue protected by NADP or by NMN; and (c) the arginine residue of the NADP-enzyme that is reactive with 1,2-cyclohexanedione with inactivation. Despite these similarities, comparison of the sequence of the NAD-enzyme with those of the other glutamate dehydrogenases of known sequences revealed relatively little overall homology as determined by computer analysis.     

Design of a disulfide-less, pharmacologically inert, and chemically competent analog of maurocalcine for the efficient transport of impermeant compounds into cells

Maurocalcine is a 33-mer peptide initially isolated from the venom of a Tunisian scorpion. It has proved itself valuable as a pharmacological activator of the ryanodine receptor and has helped the understanding of the molecular basis underlying excitation-contraction coupling in skeletal muscles. Because of its positively charged nature, it is also an innovative vector for the cell penetration of various compounds. We report a novel maurocalcine analog with improved properties: (i) the complete loss of pharmacological activity, (ii) preservation of the potent ability to carry cargo molecules into cells, and (iii) coupling chemistries not affected by the presence of internal cysteine residues of maurocalcine. We did this by replacing the six internal cysteine residues of maurocalcine by isosteric 2-aminobutyric acid residues and by adding an additional N-terminal biotinylated lysine (for a proof of concept analog) or an N-terminal cysteine residue (for a chemically competent coupling analogue). Additional replacement of a glutamate residue by alanyl at position 12 further improves the potency of these analogues. Coupling to several cargo molecules or nanoparticles are presented to illustrate the cell penetration potency and usefulness of these pharmacologically inactive analogs.     

Labeling of specific lysine residues at the active site of glutamine synthetase

Glutamine synthetase (Escherichia coli) was incubated with three different reagents that react with lysine residues, viz. pyridoxal phosphate, 5'-p-fluorosulfonylbenzoyladenosine, and thiourea dioxide. The latter reagent reacts with the epsilon-nitrogen of lysine to produce homoarginine as shown by amino acid analysis, nmr, and mass spectral analysis of the products. A variety of differential labeling experiments were conducted with the above three reagents to label specific lysine residues. Thus pyridoxal phosphate was found to modify 2 lysine residues leading to an alteration of catalytic activity. At least 1 lysine residue has been reported previously to be modified by pyridoxal phosphate at the active site of glutamine synthetase (Whitley, E. J., and Ginsburg, A. (1978) J. Biol. Chem. 253, 7017-7025). By varying the pH and buffer, one or both residues could be modified. One of these lysine residues was associated with approximately 81% loss in activity after modification while modification of the second lysine residue led to complete inactivation of the enzyme. This second lysine was found to be the residue which reacted specifically with the ATP affinity label 5'-p-fluorosulfonylbenzoyladenosine. Lys-47 has been previously identified as the residue that reacts with this reagent (Pinkofsky, H. B., Ginsburg, A., Reardon, I., Heinrikson, R. L. (1984) J. Biol. Chem. 259, 9616-9622; Foster, W. B., Griffith, M. J., and Kingdon, H. S. (1981) J. Biol. Chem. 256, 882-886). Thiourea dioxide inactivated glutamine synthetase with total loss of activity and concomitant modification of a single lysine residue. The modified amino acid was identified as homoarginine by amino acid analysis. The lysine residue modified by thiourea dioxide was established by differential labeling experiments to be the same residue associated with the 81% partial loss of activity upon pyridoxal phosphate inactivation. Inactivation with either thiourea dioxide or pyridoxal phosphate did not affect ATP binding but glutamate binding was weakened. The glutamate site was implicated as the site of thiourea dioxide modification based on protection against inactivation by saturating levels of glutamate. Glutamate also protected against pyridoxal phosphate labeling of the lysine consistent with this residue being the common site of reaction with thiourea dioxide and pyridoxal phosphate.     

Insights into the phosphoregulation of beta-secretase sorting signal by the VHS domain of GGA1

BACE (β-site amyloid precursor protein cleaving enzyme, β-secretase) is a type-I membrane protein which functions as an aspartic protease in the production of β-amyloid peptide, a causative agent of Alzheimer's disease. Its cytoplasmic tail has a characteristic acidic-cluster dileucine motif recognized by the VHS domain of adaptor proteins, GGAs (Golgi-localizing, γ-adaptin ear homology domain, ARF-interacting). Here we show that BACE is colocalized with GGAs in the trans -Golgi network and peripheral structures, and phosphorylation of a serine residue in the cytoplasmic tail enhances interaction with the VHS domain of GGA1 by about threefold. The X-ray crystal structure of the complex between the GGA1-VHS domain and the BACE C-terminal peptide illustrates a similar recognition mechanism as mannose 6-phosphate receptors except that a glutamine residue closes in to fill the gap created by the shorter BACE peptide. The serine and lysine of the BACE peptide point their side chains towards the solvent. However, phosphorylation of the serine affects the lysine side chain and the peptide backbone, resulting in one additional hydrogen bond and a stronger electrostatic interaction with the VHS domain, hence the reversible increase in affinity.     

A conservative amino acid substitution, arginine for lysine, abolishes export of a hybrid protein in Escherichia coli. Implications for the mechanism of protein secretion

A hybrid protein that comprises the beta-lactamase signal peptide fused precisely to chicken muscle triosephosphate isomerase is not secreted into the periplasm of Escherichia coli. The protein can be secreted, however, if an arginine residue at position 3 of the isomerase is replaced by either a serine or a proline residue. In contrast, replacement of a neighboring lysine residue has no effect on secretion of the protein. Furthermore, if the arginine is removed from position 3 to generate a secreted protein, but is then reintroduced in place of the neighboring lysine, the blockade to secretion is re-established. The singular effect of the arginine residue on secretion does not result from the role this residue plays in the formation or stabilization of the native isomerase structure: mutational alterations remote from the N terminus of the isomerase that prevent the proper folding of the protein do not relieve the block to secretion. The finding that an arginine residue prevents secretion while a lysine residue does not, suggests that basic residues near the mature N terminus of a secreted protein must be deprotonated if orderly export is to occur. This implies that the signal peptide along with the N-terminal portion of the mature protein partitions directly into the lipid bilayer in the course of the secretory process.     

Identification of lysine residue involved in inactivation of brain glutamate dehydrogenase isoproteins by o-phthalaldehyde

Incubation of two types of glutamate dehydrogenase (GDH) isoproteins from bovine brain with o-phthalaldehyde resulted in a time-dependent loss of enzyme activity. The inactivation was partially prevented by preincubation of the GDH isoproteins with 2-oxoglutarate or NADH. Spectrophotometric studies indicated that the inactivation of GDH isoproteins with o-phthalaldehyde resulted in isoindole derivatives characterized by typical fluorescence emission spectra with a stoichiometry of one isoindole derivative per molecule of enzyme subunit. There were no differences between the two GDH isoproteins in sensitivities to inactivation by o-phthalaldehyde indicating that the microenvironmental structures of the GDH isoproteins are very similar to each other. Tryptic peptides of the isoproteins, modified with and without protection, identified a selective modification of one lysine as in the region containing the sequence L-Q-H-G-S-I-L-G-F-P-X-A-K for both GDH isoproteins. The symbol X indicates a position for which no phenylthiohydantoin-amino acid could be assigned. The missing residue, however, can be designated as an o-phthalaldehyde-labeled lysine since the sequences including the lysine residue in question have a complete identity with those of the other mammalian GDHs. Also, trypsin was unable to cleave the labeled peptide at this site. Both amino acid sequencing and compositional analysis identified Lys-306 as the site of o-phthalaldehyde binding within the brain GDH isoproteins.     

Amino acid sequence of the N-terminal non-collagenous segment of dermatosparactic sheep procollagen type I.

The non-collagenous N-terminal segment of type I procollagen from dermatosparactic sheep skin was isolated in the form of the peptide Col 1 from a collagenase digest of the protein. The peptide has a blocked N-terminus, which was identified as pyrrolid-2-one-5-carboxylic acid. Appropriate overlapping fragments were prepared from reduced and alkylated peptide Col 1 by cleavage with trypsin at lysine, arginine and S-aminoethyl-cysteine residues and by cleavage with staphylococcal proteinase at glutamate residues. Amino acid sequence analysis of these fragments by Edman degradation and mass spectrometry established the whole sequence of peptide Col 1 except for a peptide junction (7--8) and a single Asx residue (44), and demonstrated that peptide Col 1 consists of 98 amino acid residues. The N-terminal portion of peptide Col 1 (86 residues) shows an irregular distribution of glycine, whereas the C-terminal portion (12 residues) possesses the triplet structure Gly-Xy and is apparently derived from the precursor-specific collagenous domain of procollagen. The central region of the peptide contains ten cysteine residues located between positions 18 and 73 and shows alternating polar and hydrophobic sequence elements. The regions adjacent to the cysteine-rich portion have a hydrophilic nature and are abundant in glutamic acid. The data are consistent with previous physicochemical and immunological evidence that distinct regions at the N- and C-termini of the non-collagenous domain possess a less rigid conformation than does the central portion of the molecule.     

Peptidoglycan cross-linking and teichoic acid attachment in Streptococcus pneumoniae.

Autolysin-defective pneumococci continue to synthesize both peptidoglycan and teichoic acid polymers (Fischer and Tomasz, J. Bacteriol. 157:507-513, 1984). Most of these peptidoglycan polymers are released into the surrounding medium, and a smaller portion becomes attached to the preexisting cell wall. We report here studies on the degree of cross-linking, teichoic acid substitution, and chemical composition of these peptidoglycan polymers and compare them with normal cell walls. peptidoglycan chains released from the penicillin-treated pneumococci contained no attached teichoic acids. The released peptidoglycan was hydrolyzed by M1 muramidase; over 90% of this material adsorbed to vancomycin-Sepharose and behaved like disaccharide-peptide monomers during chromatography, indicating that the released peptidoglycan contained un-cross-linked stem peptides, most of which carried the carboxy-terminal D-alanyl-D-alanine. The N-terminal residue of the released peptidoglycan was alanine, with only a minor contribution from lysine. In addition to the usual stem peptide components of pneumococcal cell walls (alanine, lysine, and glutamic acid), chemical analysis revealed the presence of significant amounts of serine, aspartate, and glycine and a high amount of alanine and glutamate as well. We suggest that these latter amino acids and the excess alanine and glutamate are present as interpeptide bridges. Heterogeneity of these was suggested by the observation that digestion of the released peptidoglycan with the pneumococcal murein hydrolase (amidase) produced peptides that were resolved by ion-exchange chromatography into two distinct peaks; the more highly mobile of these was enriched with glycine and aspartate. The peptidoglycan chains that became attached to the preexisting cell wall in the presence of penicillin contained fewer peptide cross-links and proportionally fewer attached teichoic acids than did their normal counterparts. The normal cell wall was heavily cross-linked, and the cross-linked peptides were distributed equally between the teichoic acid-linked and teichoic acid-free fragments.     

4-Aminobutyrate aminotransferase: identification of lysine residues connected with catalytic activity

Bis-PLP (P'P2-bis[5'-pyridoxal]diphosphate) was used as a probe of the catalytic site of 4-aminobutyrate aminotransferase. It reacts with lysine residues connected with aminotransferase activity and the binding of 1 mol of reduced bis-PLP/enzyme monomer abrogates catalytic activity. The reactive lysine residues are characterized by low pK values (pK = 7.3). The presence of substrate 2-oxoglutarate (4 mM) prevents inactivation of the aminotransferase treated with bis-PLP. After tryptic digestion of the enzyme modified with bis-PLP and reduced with tritiated NaBH4, a radioactive peptide absorbing at 320 nm was separated by reverse-phase high-performance liquid chromatography. The amino acid sequence of the radioactive peptide, elucidated by Edman degradation, revealed that a specific lysine residue of monomeric 4-aminobutyrate aminotransferase has reacted with bis-PLP. The sequence of the modified peptide differs from the sequence of the peptide bearing the cofactor pyridoxal-5-P covalently attached to a lysine residue. It is postulated that the modified lysine residue is involved in direct interactions with negatively charged carboxylic groups of 2-oxoglutarate.     

Identification of the lysine residue modified during the activation of acetimidylation of horse liver alcohol dehydrogenase.

A single amino group in horse liver alcohol dehydrogenase was modified with methyl(14C)acetimidate by a differential labeling procedure. Lysine residues outside the active site were modified with ethyl acetimidate while a lysine residue in the active site was protected by the formation of an enzyme-NAD+-pyrazole complex. After the protecting reagents were removed, the enzyme was treated with methyl(14C)acetimidate. Enzyme activity was enhanced 13-fold as 1.1 (14C)acetimidyl group was incorporated per active site. A labeled peptide was isolated from a tryptic-chymotryptic digest of the modified enzyme in 35% overall yield. Amino acid composition and sequential Edman degradations identified the peptide as residues 219-229; lysine residue 228 was modified with the radioactive acetimidyl group.     

Site-directed mutagenesis studies on the binding of the globular domain of linker histone H5 to the nucleosome.

The globular domain of the linker histone H5 has been expressed in Escherichia coli. The purified peptide is functional as it permits chromatosome protection during micrococcal nuclease digestion of chromatin reconstituted with the peptide, indicating that it binds correctly at the dyad axis of the nucleosomal core particle. The globular domain residue lysine 64 is highly conserved within the linker histone family, and site-directed mutagenesis has been used to assess the importance of this residue in the binding of the globular domain of linker histone H5 to the nucleosome. Recombinant peptides mutated at lysine 64 are unable to elicit chromatosome protection to the same degree as the wild-type peptide, and since they appear to be fully folded, these observations confirm a major role for this residue in determining the effective interaction between the globular domain of histone H5 and the nucleosome.     

Monkey erythropoietin gene: cloning, expression and comparison with the human erythropoietin gene

The erythropoietin (Epo) gene from Cynomolgus monkeys has been isolated from a kidney cDNA library using mixed 20-mer oligodeoxynucleotide probes. The gene encodes a 168 amino acid (aa) mature protein with a calculated Mr of 18,490 and a presumptive signal peptide of 24 aa. The Epo gene, when transfected into Chinese hamster ovary (CHO) cells, produces a glycosylated protein with an apparent Mr of 34,000. The expressed product is biologically active in vivo. The monkey gene exhibits 92% and 94% homology to the human gene at the aa and nucleotide sequence levels, respectively. When compared with the human Epo, monkey Epo has an additional 3-aa residue at the N terminus of the mature protein and a deletion of an internal lysine residue.