Primary structures of the catalytic subunits from two molecular forms of acetylcholinesterase. A comparison of NH2-terminal and active center sequences

Two distinct classes of acetylcholinesterase exist in near equal amounts in the electric organ of Torpedo californica. A globular 5.6 S form is a dimer which possesses a hydrophobic region. The second form is present as elongated species that sediment at 17 and 13 S and contain structural subunits disulfide-linked to the catalytic subunits. Removal of the structural subunits by mild proteolysis yields a tetramer of catalytic subunits which sediments at 11 S. To compare the primary structures of the catalytic subunits of the 5.6 S and 11 S forms of acetylcholinesterase, amino acid sequences from the active sites and from the amino-terminal regions have been elucidated. Active site serines were labeled with [3H]isopropyl fluorophosphate. After digestion with trypsin, the resultant peptides were resolved by elution from a size-exclusion column followed by reverse-phase high performance liquid chromatography. Each active site tryptic peptide contained 24 residues and identical sequences were found in this peptide for the 5.6 S and 11 S forms of the enzyme. The sequence flanking the active site serine revealed extensive homology with the published sequence of human serum cholinesterase as well as a lesser degree of homology with other known serine proteases and esterases. The sequences of the amino-terminal region also appear to be identical for both enzyme forms although we note variation in the ratio of Glu and Gln at position 5. The amino-terminal sequence exhibits only partial homology with the published sequence of human serum cholinesterase.     

Profile of the disulfide bonds in acetylcholinesterase

The inter- and intrasubunit disulfide bridges for the 11 S form of acetylcholinesterase isolated from Torpedo californica have been identified. Localized within the basal lamina of the synapse, the dimensionally asymmetric forms of acetylcholinesterase contain either two (13 S) or three (17 S) sets of catalytic subunits linked to collagenous and noncollagenous structural subunits. Limited proteolysis of these molecules yields a tetramer of catalytic subunits that sediments at 11 S. Each catalytic subunit contains 8 cysteine residues which were identified following tryptic digestion of the reduced, 14C-carboxymethylated protein. The tryptic peptides were purified by gel filtration followed by reverse-phase high performance liquid chromatography (HPLC) and then sequenced. The disulfide bonding profile was determined by treating the native, nonreduced 11 S form of acetylcholinesterase with a fluorescent, sulfhydryl-specific reagent, monobromobimane, prior to tryptic digestion. Peptides again were resolved by gel filtration and reverse-phase HPLC. One fluorescent cysteine-containing peptide was identified, indicating that a single sulfhydryl residue, Cys231, was present in its reduced form. Three pairs of disulfide-bonded peptides were identified. These were localized in the polypeptide chain based on the cDNA-deduced sequence of the protein and were identified as Cys67-Cys94, Cys254-Cys265, and Cys402-Cys521. Hence, three loops are found in the secondary structure. Cys572, located in the carboxyl-terminal tryptic peptide, was disulfide-bonded to an identical peptide and most likely forms an intersubunit cross-link. Since the 6 cysteine residues in acetylcholinesterase that are involved in intrachain disulfide bonds are conserved in the sequence of the homologous protein thyroglobulin, it is likely that both proteins share a common folding pattern in their respective tertiary structures. Cys231 and the carboxyl-terminal cysteine residue Cys572 are not conserved in thyroglobulin.     

Primary structure of acetylcholinesterase: implications for regulation and function

A cDNA encoding acetylcholinesterase (AChE) (EC 3.1.1.7) from Torpedo californica was isolated and from its nucleotide sequence the entire amino acid sequence of the processed protein and a portion of the leader peptide has been deduced. Approximately 70% of the tryptic peptides from the catalytic subunit of the 11 S form have been sequenced, and a comparison of the peptide sequences with the sequence inferred from the cDNA suggests that the cDNA sequence derives from mRNA for the 11 S form of the enzyme. The amino acid sequence is preceded by a hydrophobic leader peptide and contains an open reading frame encoding for 575 amino acids characteristic of a secreted globular protein. Eight cysteines, most of which are disulfide linked, are found along with four potential sites of N-linked glycosylation. The active-site serine is located at residue 200. Local homology is found with other serine hydrolases in the vicinity of the active site, but the enzyme shows striking global homology with the COOH-terminal portion of thyroglobulin. Further comparison of the amino acid sequences of the individual enzyme forms with other cDNA clones that have been isolated should resolve the molecular basis for polymorphism of the AChE species.     

Chemical identification of cysteine as palmitoylation site in a transmembrane protein (Semliki Forest virus E1)

The palmitoylation site of the membrane glycoprotein E1 of Semliki Forest virus (SFV) has been identified by chemical analysis of an acylpeptide. 3H-Palmitoylated E1 isolated from SFV grown in baby hamster kidney cells was digested with chymotrypsin and the resulting peptides subjected to high performance liquid chromatography on a wide-pore column. The 3H-acylated peptide fraction peaked at above 60% 2-propanol in the eluent, indicating its hydrophobic character. Polyacrylamide gel electrophoresis analysis revealed a molecular weight of about Mr = 6000 for the radiolabeled peptide. Manual sequencing of this material by the 4-N,N'-dimethylaminoazobenzene-4'-isothiocyanate/phenylisothiocyanate procedure on solid phase revealed the amino-terminal sequence Ala-Ala-Ser-His-Ser-Asn-Val-Val-Phe-Pro. The same peptide also labels with [35S]cysteine. Comparison with the deduced amino acid sequence of E1 revealed that the palmitoylated peptide contains at least 43 amino acid residues, and thus includes the membrane spanning region down to the only cysteine residue five positions up from the carboxyl terminus of E1. Since [3H]palmitic acid was cleaved from E1 with thiol reagents, and since the peptide labels with [14C]iodoacetamide only after the release of fatty acids by hydroxylamine treatment, cysteine in position 433 represents the palmitoylation site in SFV E1.     

In vitro poly(ADP-ribosyl)ation of seminal ribonuclease

The site of in vitro ADP-ribosylation of seminal ribonuclease was determined. Seminal enzyme was found to be a good receptor of [14C]ADP-ribose residues under the reaction conditions used. The recovery of [14C]ADP-ribosylated RNase was about 65% after purification. After tryptic digestion of modified enzyme, a fraction containing [14C]ADP-ribosylated peptides was separated from the others by ion-exchange chromatography on M82 resin. Radioactive peptides were then purified by affinity chromatography on anti-poly(ADP-ribose)IgG-Sepharose. High performance liquid chromatography of a mixture obtained after pronase digestion of purified ADP-ribosylated peptides revealed only one radioactive peptide whose amino acid composition corresponded to a peptide that has equimolar quantities of aspartic acid, serine, and glycine. Carboxypeptidase Y digestion of this peptide showed that its amino acid sequence was Asp-Ser-Gly. Only position 14-16 of seminal RNase corresponded to this sequence. The chemical stability of the ADP-ribose/enzyme linkage indicated that aspartic acid 14 is the modification site in seminal RNase.     

Acetylcholinesterase: characterization of native and proteolytically derived forms and identification of structural protein components.

The assymmetric 18S and 14S forms of acetylcholinesterase (EC 3.1.1.7) from Electrophorus electricus purified by affinity chromatography on N-methylacridinium Sepharose 2B were subjected to trypsin or collagenase proteolysis and changes in the enzyme composition and structure were monitored by sucrose gradient sedimentation, gel chromatography, and sodium dodecyl sulphate - polyacrylamide gel electrophoresis. A distinction between autolytic and tryptic degradation products is described and the generation of two new forms of acetylcholinesterase from the 18S and 14S enzyme by collagenase proteolysis is reported. The species derived from the 18S form of acetylcholinesterase has a sedimentation coefficient of 21.1S and a Stokes radius of 12.9 nm while the 14S form gives rise to a 17.3S species with a Stokes radius of 11.1 nm. The proteolytically sensitive component ('tail') of the asymmetric forms of acetylcholinesterase is identified with a subunit of 45 000 daltons on sodium dodecyl sulphate - polyacrylamide electrophoresis gels.     

Protein chemistry of the Neurospora crassa plasma membrane H+-ATPase

A highly effective procedure for fragmenting the Neurospora crassa plasma membrane H+-ATPase and purifying the resulting peptides is described. The enzyme is cleaved with trypsin to form a limit digest containing both hydrophobic and hydrophilic peptides, and the hydrophobic and hydrophilic peptides are then separated by extraction with an aqueous ammonium bicarbonate solution. The hydrophilic peptides are fractionated by Sephadex G-25 column chromatography into three pools, and the individual peptides in each pool are purified by high-performance liquid chromatography. The hydrophobic peptides are dissolved in neat trifluoroacetic acid (TFA), diluted with chloroform-methanol (1:1), and the hydrophobic peptide solution thus obtained is then fractionated by Sephadex LH-60 column chromatography in chloroform-methanol (1:1) containing 0.1% TFA. The recoveries in all of the above procedures are greater than 90%. The N-terminal amino acid sequences of three of the hydrophobic H+-ATPase peptides purified by this methodology have been determined, which establishes the position of these peptides in the 100,000 Da polypeptide chain by reference to the published gene sequence, and documents the sequencability of the hydrophobic peptides purified in this way. This methodology should facilitate the identification of a variety of amino acid residues important for the structure and function of the H+-ATPase molecule. Moreover, the overall strategy for working with the protein chemistry of the H+-ATPase should be applicable to other amphiphilic integral membrane proteins as well.     

Identification of the reactive sulfhydryl and sequences of cysteinyl-tryptic peptides from beef heart aconitase

In an accompanying paper (Kennedy, M. C., Spoto, G., Emptage, M. H., and Beinert, H. (1988) J. Biol. Chem. 263, 8190-8193), it was shown that one cysteine per mol of aconitase is modified by a variety of sulfhydryl reagents. We have identified the tryptic peptide that contains the iodoacetamide-reactive cysteine. We have also demonstrated that this cysteine is the primary site of modification by phenacyl bromide (2-bromoacetophenone), a spin label analogue of N-ethylmaleimide (HO-461) and iodoacetate in both the 3Fe and 4Fe forms of aconitase. The amino acid sequence of the peptide containing the reactive cysteine from beef heart aconitase shares no homology with the reactive cysteine-containing peptide reported for pig heart aconitase (Hahm, K.-S., Gawron, O., and Piszkiewicz, D. (1981) Biochim. Biophys. Acta 667, 457-461). We also report the amino acid compositions and sequences of seven other cysteine-containing tryptic peptides from beef heart aconitase. However, none of the cysteinyl peptides isolated were found to correspond to the reported pig heart reactive cysteinyl peptide. Evidence is also presented that no previously unreactive cysteine becomes exposed and reactive to sulfhydryl reagents in the conversion from the [4Fe-4S] cluster of the enzyme to the [3Fe-4S] cluster. We conclude from this that any potential cysteine ligand to the Fea site of the cluster must be inaccessible to solvent in the 3Fe form or, alternatively, that active 4Fe aconitase does not contain a cysteine ligand to the Fea site.     

An active serine is involved in covalent substrate amino acid binding at each reaction center of gramicidin S synthetase.

The condensing peptide forming multienzyme of gramicidin S synthetase (gramicidin S synthetase 2) was specifically labeled at its putative thiotemplate sites for L-valine and L-leucine by covalent incorporation of the 14C-labeled substrate amino acids. The thioester complexes of the multienzyme were digested with CNBr, Staphylococcus aureus V8 protease, and pepsin. Reaction center peptides containing the [14C]valine and [14C]leucine labels were isolated in pure form. They show a high degree of sequence similarity and contain the same consensus sequence LGGH/DXL. The labels were eliminated in the first Edman degradation step. A dehydroalanine was identified which can originate from either a cysteine or a serine. The comparison of the chemical results with the deduced amino acid sequence of the grsB gene encoding the gramicidin S synthetase 2 revealed that 4 such motifs are located within the gene structure, each of them being localized in the 3'-terminal region of one of 4 gene segments grsB1-B4. They have a size of approximately 2 kilobases and presumably code for the 4 amino acid activating domains of the synthetase. Surprisingly a serine was found at each putative substrate amino acid-binding position instead of a cysteine as postulated by the thiotemplate mechanism. Therefore the data suggest that active serine residues are involved in nonribosomal peptide syntheses of microbial peptides.     

Direct photoaffinity labeling of cysteine 211 or a nearby amino acid residue of beta-tubulin by guanosine 5'-diphosphate bound in the exchangeable site

Tubulin with [8-14C]GDP bound in the exchangeable site was exposed to ultraviolet light, and radiolabel was cross-linked to two peptide regions of the beta-subunit. Following enrichment for peptides cross-linked to guanosine by boronate chromatography, we confirmed that the cysteine 12 residue was the major site of cross-linking. However, significant radiolabel was also incorporated into a peptide containing amino acid residues 206 through 224. Although every amino acid in this peptide except cysteine 211 was identified by sequential Edman degradation, implying that this was the amino acid residue cross-linked to guanosine, radiolabel at C-8 was usually lost during peptide processing (probably during chromatography at pH 10). Consequently, the radiolabeled amino acid could not be unambiguously identified.     

Characterization of multiple forms of prostatropin (prostate epithelial cell growth factor) from bovine brain

Two molecular forms of prostatropin distributed among five chromatographic peaks have been isolated from bovine brain by heparin-Sepharose affinity and reverse phase high performance liquid chromatography. One form has an apparent molecular weight of 16000 and an amino terminal sequence of asn-tyr-lys-lys-pro-lys-leu-leu-tyr-x-ser-asn-gly. The other form has an apparent molecular weight of 18000 and a blocked amino terminus. Both forms are similar in amino acid composition. The sequence of a proteolytic fragment from the blocked form overlaps the NH2-terminal sequence of the unblocked form, therefore, the smaller form may be derived from the larger form through proteolytic processing. Both forms contain regions identical in sequence to brain-derived, heparin-binding growth factors that have been isolated on the basis of mitogenic activity for fibroblasts and endothelial cells. Both forms of prostatropin exhibit potent mitogenic activity for normal and tumor prostate epithelial cells.     

Complete amino acid sequence of NADH-cytochrome b5 reductase purified from human erythrocytes

The complete amino acid sequence of soluble NADH-cytochrome b5 reductase purified from human erythrocytes was determined. The enzyme, which contained 8 methionine residues, was cleaved by cyanogen bromide. The resulting nine peptides were separated by gel filtration and purified further by high-performance liquid chromatography. The purified peptides were sequenced by automated Edman degradation. Three large CNBr peptides, residues 1-101, 109-151, and 169-231, were further fragmented with trypsin, Staphylococcus aureus V8 protease or a lysyl endopeptidase of Achromobacter lyticus. The peptides obtained from the tryptic digest of citraconylated FAD-depleted apoprotein completed the alignments of the other peptides. The enzyme was composed of 275 amino acid residues. The 4 functionally important cysteine residues were located in the COOH-terminal portion. The molecular weight of the protein was calculated to be 31,260 without FAD. A prediction of the secondary structure was made by the method of Chou and Fasman. The protein was hydrophilic as a whole (43% polarity), but some regions were rich in hydrophobic residues. From the sequence homology of this enzyme with the pyridine nucleotide-binding sites of other flavoproteins, three candidates for the FAD and NADH-binding domains were suggested.     

Identification of insulin intermediates and sites of cleavage of native insulin by insulin protease from human fibroblasts

We have studied the time sequence degradation of native insulin by insulin protease from human fibroblast using multiple steps involving purification of the products by high performance liquid chromatography, determination of peak composition by amino acid sequence analysis, and confirmation of structure by mass spectrometry and thus elucidated the sites of cleavage of insulin by human insulin protease. We observed that as early as 0.5 min of incubation, three major new peptide peaks, intact insulin, and four smaller peptide peaks can be detected. The major peptides are portions of the insulin molecule, with the amino ends of the A and B chains or the carboxyl ends of the A and B chains still connected by disulfide bonds. Peptide peak I is A1-13-B1-9. Peptide peak II is A1-14-B1-9. Peptide peak III is A14-21-B14-30. The smaller peptide peaks are A14-21-B17-30, A15-21-B14-30, A15-21-B10-30, and A14-21-B10-30. The major peptide bond cleavage sites therefore consist of A13-14, A14-15, B9-10, B13-14, and B10-17. With longer incubation times, peptide peak II appears to lose the A14 tyrosine to form peptide peak I. This peptide I, which is the amino end of the A and B chains, is not further degraded even after 1.5 h of incubation. With longer incubation times, the peptides containing the carboxyl ends of the A and B chains are further degraded to form products from cleavage at the A18-19, B14-15, B25-26, and a small amount of A19-20, B10-11, and B24-25 cleavage and the emergence of 2-5-amino acid peptide chains, tyrosine, alanine, histidine, and leucine-tyrosine. We conclude, based on the three-dimensional structure of insulin, that human insulin protease recognizes the alpha-helical regions around leucine-tyrosine bonds and that final degradation steps to small peptides do not require lysosomal involvement.     

The electric organ ofDiscopyge tschudii: Its innervated face and the biology of acetylcholinesterase

An ultrastructural, histochemical, and biochemical study of the electric organ of the South American Torpedinid ray, Discopyge tschudii, was carried out. Fine structural cytochemical localization of acetylcholinesterase (AChE) indicated that most of the esterase was associated with the basal lamina. Electron microscopy indicated no marked differences in the electrocyte ultrastructure between Discopyge and Torpedo californica. Discopyge electric organ possessed three molecular forms, two asymmetric forms (16 S and 13 S) and one globular hydrophobic form (6.5 S). The asymmetric 16 S AChE form was solubilized by heparin, a sulfated glycosaminoglycan, suggesting that heparin-like macromolecules are involved in the binding of the enzyme to the basal lamina. Our results show that cell-free translated AChE peptides, synthesized using Discopyge electric organ poly(A+) RNA, correspond to a main band of 62,000 daltons which probably represents the catalytic subunit of the asymmetric AChE.     

Isolation and characterisation of an unexpected class of insulinotropic peptides in the skin of the frog Agalychnis litodryas

Skin secretions of the frog Agalychnis litodryas were evaluated for the isolation and characterisation of novel insulinotropic peptides. Crude secretions obtained from young adult frogs by mild electrical stimulation of the dorsal skin surface were purified by reverse-phase high-performance liquid chromatography (HPLC) yielding 70 fractions. In acute 20-min incubations with glucose responsive BRIN-BD11 cells, fractions 39-42 (band 1) and fractions 44-46 (band 2) significantly stimulated insulin release by 2-3.5-fold compared with 5.6 mM glucose alone. Pooled fractions in band 1 and band 2 were rechromatographed to reveal 20 homogenous peptide peaks, which elicited significant 1.5-4-fold increases in insulin release. Mass spectrometry analyses indicated molecular masses of between 1649.2 and 4988.9 Da. The two peptides with the greatest insulin-releasing activity were directly subjected to N-terminal amino acid sequence analysis. The sequence of the 3020 Da peptide, called frog skin insulinotropic peptide or FSIP, was determined as AVWKDFLKNIGKAAGKAVLNSVTDMVNE, which has 79% homology with the C-terminal of the 75 amino acid dermaseptin BIV precursor. A partial N-terminal sequence was determined for the 2546.2 Da peptide as MLADVFEKIMGD... These data indicate that the skin secretions of A. litodryas frogs contain biologically active peptides which merit further evaluation as a new class of insulin secretagogues.     

Identification of a highly conserved domain in the EcoRII methyltransferase which can be photolabeled with S-adenosyl-L-[methyl-3H]methionine. Evidence for UV-induced transmethylation of cysteine 186

DNA methyltransferases can be photolabeled with S-adenosyl-L-methionine (AdoMet). Specific incorporation of radioactivity has been demonstrated after photolabeling with either [methyl-3H]AdoMet or [35S]AdoMet (Som, S., and Friedman, S. (1990) J. Biol. Chem. 265, 4278-4283). The labeling is believed to occur at the AdoMet binding site. With the purpose of localizing the site responsible for [methyl-3H]AdoMet photolabeling, we cleaved the labeled EcoRII methyltransferase by chemical and enzymatic reactions and isolated the radiolabeled peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure liquid chromatography. The labeled peptides were identified by amino-terminal sequencing. A common region was localized which accounted for 65-70% of the total label. This region includes a highly conserved core sequence present in all DNA (cytosine 5)-methyltransferases. One such fragment was digested further with chymotrypsin, and amino acid analysis of the resulting 3H-labeled peptide was consistent with the sequence Ala-Gly-Phe-Pro-(Cys)-Gln-Pro-Phe-Ser-Leu. However, the cysteine residue was not recovered as carboxymethylcysteine. The Pro-Cys bond was found to be protected from cleavage at cysteine residues after cyanylation. These results suggest that the cysteine residue is modified by the labeling reaction. The chymotryptic fragment was hydrolyzed enzymatically to single amino acids, and the labeled amino acid was identified as S-methylcysteine by thin layer chromatography. These results indicate that the cysteine residue is located at or close to the AdoMet binding site of EcoRII methyltransferase.     

Origin of bombesin-like peptides in human fetal lung

Four different forms of bombesin-like immunoreactive peaks were detected in extracts of human fetal lung by the use of reversed-phase high performance liquid chromatography (HPLC). Peaks I, II, III and IV, (increasing retention time), were eluted using a 14-38% of acetonitrile gradient containing 0.1% trifluoroacetic acid (TFA). Peak II was the major material found in the extract of human fetal lung obtained at 16-20 weeks gestation. None of the four compounds contained in the eluted peaks had the same retention time as amphibian bombesin or porcine gastrin releasing peptide (GRP). On reversed-phase HPLC using two different solvent systems TFA or heptafluorobutyric acid (HFBA) as a hydrophobic counter ion, and in gel filtration chromatography, the chromatographic behavior of the main peak (peak II) was the same as that of the carboxyl terminal fragments of GRP, GRP18-27 or GRP19-27. This suggested that the peptide(s) in peak II resembled in composition the carboxy terminal 9 or 10 amino acids of porcine GRP. Following tryptic digestion the material in peak IV was converted to the more polar compound present in peak II. Two other peptide peaks were eluted close to peak II and these were presumed to be a modification of this main peak. One of the possible biosynthetic steps in the formation of bombesin-like peptides in human fetal lung could be a tryptic conversion of a less polar peptide to a more polar form (peak IV to II).     

Degradation of a signal peptide by protease IV and oligopeptidase A.

The degradation of the prolipoprotein signal peptide in vitro by membranes, cytoplasmic fraction, and two purified major signal peptide peptidases from Escherichia coli was followed by reverse-phase liquid chromatography (RPLC). The cytoplasmic fraction hydrolyzed the signal peptide completely into amino acids. In contrast, many peptide fragments accumulated as final products during the cleavage by a membrane fraction. Most of the peptides were similar to the peptides formed during the cleavage of the signal peptide by the purified membrane-bound signal peptide peptidase, protease IV. Peptide fragments generated during the cleavage of the signal peptide by protease IV and a cytoplasmic enzyme, oligopeptidase A, were identified from their amino acid compositions, their retention times during RPLC, and knowledge of the amino acid sequence of the signal peptide. Both enzymes were endopeptidases, as neither dipeptides nor free amino acids were formed during the cleavage reactions. Protease IV cleaved the signal peptide predominantly in the hydrophobic segment (residues 7 to 14). Protease IV required substrates with hydrophobic amino acids at the primary and the adjacent substrate-binding sites, with a minimum of three amino acids on either side of the scissile bond. Oligopeptidase A cleaved peptides (minimally five residues) that had either alanine or glycine at the P'1 (primary binding site) or at the P1 (preceding P'1) site of the substrate. These results support the hypothesis that protease IV is the major signal peptide peptidase in membranes that initiates the degradation of the signal peptide by making endoproteolytic cuts; oligopeptidase A and other cytoplasmic enzymes further degrade the partially degraded portions of the signal peptide that may be diffused or transported back into the cytoplasm from the membranes.     

Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 5 from the dacA mutant strain of Escherichia coli (TMRL 1222)

The localization of the active site of penicillin-binding protein 5 from the dacA mutant of Escherichia coli strain TMRL 1222 has been determined. The protein was purified to homogeneity and labeled with [14C] penicillin G. The labeled protein was digested with trypsin, and the active site tryptic peptide was purified by a combination of gel filtration and high-pressure liquid chromatography. Sequencing of the purified [14C]penicilloyl peptide yielded the sequence Arg-Asp-Pro-Ala-Ser-Leu-Thr-Lys, which corresponds to residues 40-47 of the gene sequence (Broome-Smith, J., Edelman, A., and Spratt, B. G. (1983) in The Target of Penicillin (Hakenbeck, R., Holtje, J.-V., and Labischinski, H., eds) pp. 403-408, Walter de Gruyter, Berlin). The catalytic amino acid residue that forms a covalent bond with penicillin was identified by treating the purified [14C]penicilloyl peptide with a mixture of proteases and then separating the radioactive products using high-pressure liquid chromatography. Analysis of the radioactive peaks by amino acid analysis confirmed that it is the serine residue that reacts with the beta-lactam ring of penicillin.     

Mechanism of the irreversible inactivation of mouse ornithine decarboxylase by alpha-difluoromethylornithine. Characterization of sequences at the inhibitor and coenzyme binding sites.

Mouse ornithine decarboxylase (ODC) was expressed in Escherichia coli and the purified recombinant enzyme used for determination of the binding site for pyridoxal 5'-phosphate and of the residues modified in the inactivation of the enzyme by the enzyme-activated irreversible inhibitor, alpha-difluoromethylornithine (DFMO). The pyridoxal 5'-phosphate binding lysine in mouse ODC was identified as lysine 69 of the mouse sequence by reduction of the purified holoenzyme form with NaB[3H]4 followed by digestion of the carboxymethylated protein with endoproteinase Lys-C, radioactive peptide mapping using reversed-phase high pressure liquid chromatography and gas-phase peptide sequencing. This lysine is contained in the sequence PFYAVKC, which is found in all known ODCs from eukaryotes. The preceding amino acids do not conform to the consensus sequence of SXHK, which contains the pyridoxal 5'-phosphate binding lysine in a number of other decarboxylases including ODCs from E. coli. Using a similar procedure to analyze ODC labeled by reaction with [5-14C]DFMO, it was found that lysine 69 and cysteine 360 formed covalent adducts with the inhibitor. Cysteine 360, which was the major adduct accounting for about 90% of the total labeling, is contained within the sequence -WGPTCDGL(I)D-, which is present in all known eukaryote ODCs. These results provide strong evidence that these two peptides form essential parts of the catalytic site of ODC. Analysis by fast atom bombardment-mass spectrometry of tryptic peptides containing the DFMO-cysteine adduct indicated that the adduct formed in the enzyme was probably the cyclic imine S-(2-(1-pyrroline)methyl)cysteine. This is readily oxidized to S-((2-pyrrole)methyl)cysteine or converted to S-((2-pyrrolidine)methyl)cysteine by NaBH4 reduction. This adduct is consistent with spectral evidence showing that inactivation of the enzyme with DFMO does not entail the formation of a stable adduct between the pyridoxal 5'-phosphate, the enzyme, and the inhibitor.