Evidence for two lipoic acid residues per lipoate acetyltransferase chain in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The reaction of two maleimides, N-ethylmaleimide and bis-(N-maleimidomethyl) ether, with the pyruvate dehydrogenase multienzyme complex of Escherichia coli in the presence of the substrate, pyruvate, was examined. In both cases, the reaction was demonstrated to be almost exclusively with the lipoate acetyltransferase component, and evidence is presented to show that the most likely sites of reaction are the lipoic acid residues covalently bound to this component. With both reagents the stoicheiometry of the reaction was measured: 2 mol of reagent reacted with each polypeptide chain of lipoate acetyltransferase, implying that each chain bears two functionally active lipolic acid residues. This observation can be reconciled with previous determinations of the lipoic acid content of the complex by allowing for the variability of the subunit polypeptide-chain ratio that can be demonstrated for this multimeric enzyme.     

Limited proteolysis and proton NMR spectroscopy of Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex

The pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was treated with chymotrypsin at pH 7 and 0 degrees C. Loss of the overall catalytic activity lagged behind the rapid cleavage of the lipoate acetyltransferase polypeptide chains, whose apparent Mr fell from 57 000 to 45 000 as judged by sodium dodecylsulphate/polyacrylamide gel electrophoresis. The inactive chymotrypsin-treated enzyme had lost the lipoic-acid-containing regions of the lipoate acetyltransferase chains, yet remained a highly assembled structure. Treatment of this chymotryptic core complex with trypsin at pH 7.0 and 0 degrees C caused a further shortening of the lipoate acetyltransferase polypeptide chains to an apparent Mr of 28 000 and was accompanied by disassembly of the complex. The lipoic-acid-containing regions are therefore likely to be physically exposed in the intact complex, protruding from the structural core formed by the lipoate acetyltransferase component between the subunits of the other component enzymes. Proton nuclear magnetic resonance spectroscopy demonstrated that the enzyme complex contains large regions of polypeptide chain with remarkable intramolecular mobility, most of which were retained after excision of the lipoic-acid-containing regions with chymotrypsin. It is likely that the highly mobile regions are in the lipoate acetyltransferase component and facilitate movement of the lipoic acid residues. Such polypeptide chain mobility provides the molecular basis of a novel system of active-site coupling in the 2-oxo acid dehydrogenase multienzyme complexes.     

Lipoic acid is the site of substrate-dependent acetylation of component X in ox heart pyruvate dehydrogenase multienzyme complex

The recently characterized Mr-50000 polypeptide associated with mammalian pyruvate dehydrogenase complex, referred to as component or protein X, was shown to incorporate N-ethylmaleimide only in the presence of pyruvate or NADH. Component X, modified with N-ethyl[2,3-14C]maleimide in the presence of pyruvate, was isolated and subjected to acid hydrolysis. The radioactive products were resolved on an amino acid analyser and these coeluted with products from similarly modified and hydrolysed lipoate acetyltransferase. Preincubation of pyruvate dehydrogenase complex with pyruvate or NADH and acetyl-CoA resulted in a time-dependent diminution of incorporation of radiolabelled N-ethylmaleimide into component X and lipoate acetyltransferase and, correspondingly, in the extent of inhibition of overall complex activity by N-ethylmaleimide.     

Kinetic analysis of the role of lipoic acid residues in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

The catalytic roles of the two reductively acetylatable lipoic acid residues on each lipoate acetyltransferase chain of the pyruvate dehydrogenase complex of Escherichia coli were investigated. Both lipoyl groups are reductively acetylated from pyruvate at the same apparent rate and both can transfer their acetyl groups to CoASH, part-reactions of the overall complex reaction. The complex was treated with N-ethylmaleimide in the presence of pyruvate and the absence of CoASH, conditions that lead to the modification and inactivation of the S-acetyldihydrolipoic acid residues. Modification was found to proceed appreciably faster than the accompanying loss of enzymic activity. The kinetics of the modification were fitted best by supposing that the two lipoyl groups react with the maleimide at different rates, one being modified at approximately 3.5 times the rate of the other. The loss of complex activity took place at a rate approximately equal to that calculated for the modification of the more slowly reacting lipoic acid residue. The simplest interpretation of this result is that only this residue is essential in the overall catalytic mechanism, but an alternative explanation in which one lipoic acid residue can take over the function of another was not ruled out. The kinetics of inactivation could not be reconciled with an obligatory serial interaction between the two lipoic acid residues. Similar experiments with the fluorescent N-[p-(benzimidazol-2-yl)phenyl]maleimide supported these conclusions, although the modification was found to be less specific than with N-ethylmaleimide. The more rapidly modified lipoic acid residue may be involved in the system of intramolecular transacetylation reactions that couple active sites in the lipoate acetyltransferase component.     

Polypeptide-chain stoicheiometry and lipoic acid content of the pyruvate dehydrogenase complex of Escherichia coli.

The pyruvate dehydrogenase multienzyme complex was isolated from Escherichia coli grown in the presence of [35S]sulphate. The three component enzymes were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the molar ratios of the three polypeptide chains were determined by measurement of the radioactivity in each band. The chain ratio of lipoamide dehydrogenase to lipoate acetyltransferase approached unity, but there was a molar excess of chains of the pyruvate decarboxylase component. The 35S-labelled complex was also used in a new determination of the total lipoic acid content. It was found that each polypeptide chain of the lipoate acetyltransferase component appears to bear at least three lipoyl groups.     

Component X of mammalian pyruvate dehydrogenase complex: structural and functional relationship to the lipoate acetyltransferase (E2) component

The lipoate acetyltransferase (E2, Mr 70,000) and protein X (Mr 51,000) subunits of the bovine pyruvate dehydrogenase multienzyme complex (PDC) core assembly are antigenically distinct polypeptides. However comparison of the N-terminal amino acid sequence of the E2 and X polypeptides reveals significant homology between the two components. Selective tryptic release of the 14C-labelled acetylated lipoyl domains of E2 and protein X from native PDC generates stable, radiolabelled 34 and 15 kDa fragments, respectively. Thus, in contrast to E2 which contains two tandemly-arranged lipoyl domains, protein X appears to contain only a single lipoyl domain located at its N-terminus.     

Identification and analysis of novel amino acid sequence repeats and domains in Pyrobaculum aerophilum using computational tools

We have identified four repeats and five domains that are novel in proteins encoded by the Pyrobaculum aerophilum str. IM2 proteome using automated in silico methods. A "repeat" corresponds to a region comprising less than 55 amino acid residues that occurs more than once in the protein sequence and sometimes present in tandem. A "domain" corresponds to a conserved region comprising greater than 55 amino acid residues and may be present as single or multiple copies in the protein sequence. These correspond to (1) 85 amino acid residues AAG domain, (2) 72 amino acid residues GFGN domain, (3) 43 amino acid residues KGG repeat, (4) 25 amino acid residues RWE repeat, (5) 25 amino acid residues RID repeat, (6) 108 amino acid residues NDFA domain, (7) 140 amino acid residues VxY domain, (8) 35 amino acid residues LLPN repeat and (9) 98 amino acid residues GxY domain. A repeat or domain is characterized by specific conserved sequence motifs. We discuss the presence of these repeats and domains in proteins from other genomes and their probable secondary structure.     

Deduced amino acid sequence of Escherichia coli adenosine deaminase reveals evolutionarily conserved amino acid residues: implications for catalytic function

The goal of the research reported here is to identify evolutionarily conserved amino acid residues associated with enzymatic deamination of adenosine. To do this, we isolated molecular clones of the Escherichia coli adenosine deaminase gene by functional complementation of adenosine deaminase deficient bacteria and deduced the amino acid sequence of the enzyme from the nucleotide sequence of the gene. Nucleotide sequence analysis revealed the presence of a 996-nucleotide open reading frame encoding a protein of 332 amino acids having a molecular weight of 36,345. The deduced amino acid sequence of the E. coli enzyme has approximately 33% identity with those of the mammalian adenosine deaminases. With conservative amino acid substitutions the overall sequence homology approaches 50%, suggesting that the structures and functions of the mammalian and bacterial enzymes are similar. Additional amino acid sequence analysis revealed specific residues that are conserved among all three adenosine deaminases and four AMP deaminases for which sequence information is currently available. In view of previously published enzymological data and the conserved amino acid residues identified in this study, we propose a model to account for the enzyme-catalyzed hydrolytic deamination of adenosine. Potential catalytic roles are assigned to the conserved His 214, Cys 262, Asp 295, and Asp 296 residues of mammalian adenosine deaminases and the corresponding conserved amino acid residues in bacterial adenosine deaminase and the eukaryotic AMP deaminases.     

Amino acid sequence of calmodulin from wheat germ

The complete amino acid sequence of calmodulin from wheat germ was determined by isolating and sequencing the cyanogen bromide and tryptic peptides. The protein consisted of 149 amino acid residues and its amino(N)-terminus was blocked with an acetyl group. Wheat germ calmodulin lacked tryptophan and contained 1 mol each of histidine, tyrosine, cysteine, and N epsilon-trimethyllysine residues per mol of the protein. A comparison of its amino acid sequence with that of bovine brain calmodulin indicated that there were eleven amino acid subsitutions other than amide assignments, two insertions and one deletion of amino acid residues in wheat germ calmodulin.     

The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A

The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A subunit I has been determined by automated Edman degradation of the cyanogen bromide fractions derived from the precursor protein. The activation peptide contains 94 amino acid residues in a unique sequence which precedes directly the amino-terminal alanine residue of carboxypeptidase A alpha. A notable feature of the activation peptide is the presence of acidic amino acid residues immediately preceding the site of activation. The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A shows extensive similarity to those of the corresponding porcine and rat enzymes.     

Kinetics and specificity of reductive acylation of lipoyl domains from 2-oxo acid dehydrogenase multienzyme complexes

Lipoamide and a peptide, Thr-Val-Glu-Gly-Asp-Lys-Ala-Ser-Met-Glu lipoylated on the N6-amino group of the lysine residue, were tested as substrates for reductive acetylation by the pyruvate decarboxylase (E1p) component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli. The peptide has the same amino acid sequence as that surrounding the three lipoyllysine residues in the lipoate acetyltransferase (E2p) component of the native enzyme complex. Lipoamide was shown to be a very poor substrate, with a Km much higher than 4 mM and a value of kcat/Km of 1.5 M-1.s-1. Under similar conditions, the three E2p lipoyl domains, excised from the pyruvate dehydrogenase complex by treatment with Staphylococcus aureus V8 proteinase, could be reductively acetylated by E1p much more readily, with a typical Km of approximately 26 microM and a typical kcat of approximately 0.8 s-1. The value of kcat/Km for the lipoyl domains, approximately 3.0 x 10(4) M-1.s-1, is about 20,000 times higher than that for lipoamide as a substrate. This indicates the great improvement in the effectiveness of lipoic acid as a substrate for E1p that accompanies the attachment of the lipoyl group to a protein domain. The free E2o lipoyl domain was similarly found to be capable of being reductively succinylated by the 2-oxoglutarate decarboxylase (E1o) component of the 2-oxoglutarate dehydrogenase complex of E. coli. The 2-oxo acid dehydrogenase complexes are specific for their particular 2-oxo acid substrates. The specificity of the E1 components was found to extend also to the lipoyl domains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bovine kidney pyruvate dehydrogenase complex. Limited proteolysis and molecular structure of the lipoate acetyltransferase component

1. Bovine kidney pyruvate dehydrogenase multienzyme complex is inactivated by elastase in a similar manner as described earlier for papain. The core component, lipoate acetyltransferase, is cleaved by elastase into an active fragment (Mr 26000) and a fragment with apparent Mr of 45000 as analyzed by dodecylsulfate gel electrophoresis. Due to the fragmentation of the core, the enzyme complex is disassembled into its component enzymes which retain their complete enzymatic activities as assayed separately. 2. A different mechanism was found for the inactivation of pyruvate dehydrogenase complex with trypsin and some other proteases (chymotrypsin, clostripain). In these cases, the pyruvate dehydrogenase component is inactivated rapidly by limited proteolysis. More slowly, the enzyme complex is disassembled simultaneously with fragmentation of the lipoate acetyltransferase which again results in an active fragment of Mr 26000 and another fragment of apparent Mr 45000. Upon prolonged proteolysis, the latter fragment is cleaved further to give products of Mr 36000 or lower. 3. The enzyme-bound lipoyl residues of the pyruvate dehydrogenase complex have been labelled covalently by incubation with [2-14C]pyruvate. After treatment of this [14C]acetyl-enzyme with papain, elastase, or trypsin, radioactivity was associated exclusively with the 45000-Mr and 36000-Mr fragments but not with the active 26000-Mr fragment. 4. It is concluded that the bovine kidney lipoate acetyltransferase core is composed of 60 subunits each consisting of two dissimilar folding domains. One of these contains the intersubunit binding sites as well as the active center for transacylation whereas the other possesses the enzyme-bound lipoyl residues.     

The complete amino acid sequence of yeast phosphoglycerate kinase.

The complete amino acid sequence of yeast phosphoglycerate kinase, comprising 415 residues, was determined. The sequence of residues 1-173 was deduced mainly from nucleotide sequence analysis of a series of overlapping fragments derived from the relevant portion of a 2.95-kilobase endonuclease-HindIII-digest fragment containing the yeast phosphoglycerate kinase gene. The sequence of residues 174-415 was deduced mainly from amino acid sequence analysis of three CNBr-cleavage fragments, and from peptides derived from these fragments after digestion by a number of proteolytic enzymes. Cleavage at the two tryptophan residues with o-iodosobenzoic acid was also used to isolate fragments suitable for amino acid sequence analysis. Determination of the complete sequence now allows a detailed interpretation of the existing high-resolution X-ray-crystallographic structure. The sequence -Ile-Ile-Gly-Gly-Gly- occurs twice in distant parts of the linear sequence (residues 232-236 and 367-371). Both these regions contribute to the nucleoside phosphate-binding site. A comparison of the sequence of yeast phosphoglycerate kinase reported here with the sequences of phosphoglycerate kinase from horse muscle and human erythrocytes shows that the yeast enzyme is 64% identical with the mammalian enzymes. The yeast has strikingly fewer methionine, cysteine and tryptophan residues.     

Purification and amino acid sequence of a bacteriocin produced by Pediococcus acidilactici.

A bacteriocin produced by Pediococcus acidilactici has been purified to homogeneity by a rapid and simple four-step purification procedure which includes ammonium sulphate precipitation, chromatography with a cation-exchanger and Octyl Sepharose, and reverse-phase chromatography. The purification resulted in an approximately 80,000-fold increase in the specific activity and about a 6-fold increase in the total activity. The amino acid composition and sequencing data indicated that the bacteriocin contained 43-44 amino acid residues. The predicted M(r) and isolectric point of the bacteriocin are about 4600 and 8.6, respectively. Comparing the amino acid sequence of this bacteriocin with the sequences of leucocin A-UAL 187, sakacin P and curvacin A (bacteriocins produced by Leuconostoc gelidum, Lactobacillus sake and Lactobacillus curvatus, respectively) revealed that all four bacteriocins had in their N-terminal region the sequence Tyr-Gly-Asn-Gly-Val-Xaa-Cys, indicating that this concensus sequence is of fundamental importance for this group of bacteriocins. The bacteriocin from P. acidilactici and sakacin P were very similar, having at least 25 common amino acid residues. The sequence similarity was greatest in the N-terminal half of the molecules--17 of the first 19 residues were common--indicating the fundamental importance of this region. Leucocin A-UAL 187 and curvacin A had, respectively, at least 16 and 13 amino acid residues in common with the bacteriocin from P. acidilactici.     

Two lipoyl domains in the dihydrolipoamide acetyltransferase chain of the pyruvate dehydrogenase multienzyme complex of Streptococcus faecalis

A fragment of DNA incorporating the gene, pdhC, that encodes the dihydrolipoamide acetyltransferase (E2) chain of the pyruvate dehydrogenase multienzyme complex of Streptococcus faecalis was cloned and a DNA sequence of 2360 bp was determined. The pdhC gene (1620 bp) corresponds to an E2 chain of 539 amino acid residues, Mr 56,466, comprising two lipoyl domains, a peripheral subunit-binding domain and an acetyltransferase domain, linked together by regions of polypeptide chain rich in alanine, proline and charged amino acids. The S. faecalis E2 chain differs in the number of its lipoyl domains from the E2 chains of all bacterial pyruvate dehydrogenase complexes hitherto described.     

Amino acid sequence of rhizopuspepsin isozyme pI 5

The complete amino acid sequence of an aspartic protease from Rhizopus chinensis, rhizopuspepsin isozyme pI 5, has been determined. Partial sequences were first obtained from the isolated isozyme by a combination of chemical and proteolytic enzyme cleavages, peptide purifications, and Edman degradations. About one-half of the sequence was revealed by this approach. To complete the amino acid sequence, a cDNA library of R. chinensis in pBR322 was constructed. An oligonucleotide probe was synthesized based on the sequence Trp-Trp-Gly-Ile-Thr, and about 40 positive clones were identified by colony hybridization. A clone, 33E2, which had an insert size of about 1.1 kilobase pairs, was found to contain the entire coding region of rhizopuspepsin isozyme pI 5. The sequence of rhizopuspepsin contains 325 amino acid residues. The alignment of the rhizopuspepsin sequence against other aspartic proteases revealed expected homology, with the closest similarity to penicillopepsin which shares 39% identical residues. Porcine pepsin shares about 36% identical residues with rhizopuspepsin.     

Isolation and sequencing of a cDNA clone encoding acid phosphatase in rat liver lysosomes

A full length cDNA for acid phosphatase in rat liver lysosomes was isolated and sequenced. The predicted amino acid sequence comprises 423 residues (48,332 Da). A putative signal peptide of 30 residues is followed by the NH2-terminal sequence of lysosomal acid phosphatase (45,096 Da). The deduced NH2-terminal 18-residue sequence is identical with that determined directly for acid phosphatases purified from the rat liver lysosomal membranes. The primary structure deduced for acid phosphatase contains 9 potential N-glycosylation sites and a hydrophobic region which could function as a transmembrane domain. It exhibits 89% and 67% sequence similarities in amino acids and nucleic acids, respectively, to human lysosomal acid phosphatase. The amino acid sequence of the putative transmembrane segment shows a complete similarity to that of the human enzyme. Northern blot hybridization analysis identified a single species of acid phosphatase mRNA (2.2 kbp in length) in rat liver.     

Amino acid sequence of the hemerythrin alpha subunit from Lingula unguis.

The amino acid sequence of the alpha subunit of the allosteric hemerythrin from Lingula unguis was determined. It consists of 117 amino acid residues. Compared with other non-allosteric hemerythrins consisting of identical subunits of 113 amino acid residues, this protein has the deletion of the N-terminal amino acid and the insertion of five amino acids in the same region as in the case of the monomeric myoerythrin from Themiste zostericola. As the amino acid sequence of the beta subunit has also been determined [Yano, H., Satake, K., Ueno, Y., & Tsugita, A. Protein Sequence and Data Analysis, in press], the complete sequence analysis of an allosteric hemerythrin has been accomplished for the first time. The difference in the octameric structures of allosteric and non-allosteric hemerythrins are discussed.     

The amino acid sequence of human beta-microseminoprotein

The complete amino acid sequence of beta-microseminoprotein of human seminal plasma was determined by automated Edman degradation of the protein and peptides which were obtained by enzymatic cleavage with trypsin, chymotrypsin and Staphylococcus aureus V8 proteinase. The carboxyl-terminal sequence of the protein was established with the aid of carboxypeptidase A. The amino acid sequence of this protein proved to be as follows: (sequence; see text) Thus, beta-microseminoprotein consisting of 93 amino acid residues has a molecular mass of 10 652 Da. The linear structure of this protein represents the first complete amino acid sequence of a sperm-coating protein specific to human seminal plasma.