The amino acid sequence encompassing the active-site histidine residue of lipoamide dehydrogenase from Escherichia coli labelled with a bifunctional arsenoxide

1986 ◽  
Vol 64 (6) ◽  
pp. 509-514 ◽  
Author(s):  
Charles F. B. Holmes ◽  
Kenneth J. Stevenson
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
High Performance ◽  
Thiamine Pyrophosphate ◽  
Initial Reaction ◽  
Cnbr Cleavage ◽  
Lipoamide Dehydrogenase ◽  
Active Site Histidine ◽  
Inhibited Enzyme

Pyruvate dehydrogenase multienzyme complex (PD complex) in the presence of pyruvate, thiamine pyrophosphate, coenzyme A, and Mg2+ (or NADH) was irreversibly inhibited with the radiolabelled bifunctional arsenoxide p-[(bromoacetyl)-amino]phenyl arsenoxide (BrCH214CONHPhAsO). The initial reaction of the reagent was with a reduced lipoyl group of the lipoamide acetyltransferase component to form a dithioarsinite complex. Following the normal catalytic reactions, the anchored reagent was delivered into the active site of the lipoamide dehydrogenase (E3) component where an irreversible alkylation ensued via the bromoacetamidyl moiety. Treatment with 2,3-dithiopropanol (to break dithioarsinite bonds) caused the radio-labelled reagent to reside with E3. E3 was isolated from the inhibited PD complex and CNBr cleavage of the inhibited enzyme yielded a single radiolabelled peptide that was purified on a cyanopropyl silica column using high performance liquid chromatography. The radiolabelled amino acid was identified (after acid hydrolysis) as N3-[14C]carboxymethyl histidine in agreement with earlier studies. The radiolabel was located in residue 14 of the peptide for which the sequence was determined as[Formula: see text]This sequence agrees with the amino acid sequence determined from the gene sequence of E3. The histidine alkylated in the E3 component of the PD complex by BrCH214CONHPhAsO is residue-444 and further establishes its active site role.

scholarly journals An amino acid sequence in the active site of lipoamide dehydrogenase from pig heart

1974 ◽  
Vol 137 (3) ◽  
pp. 505-512 ◽  
Author(s):  
Joseph P. Brown ◽  
Richard N. Perham
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Amino Acid Sequences ◽  
Disulphide Bridge ◽  
Similar Sequence ◽  
Bacterial Enzyme ◽  
Oxoglutarate Dehydrogenase ◽  
Lipoamide Dehydrogenase ◽  
Polypeptide Chains

1. The two cysteine residues forming the disulphide bridge that comprises part of the active site of lipoamide dehydrogenase from pig heart were specifically labelled with iodo[2-14C]acetic acid. 2. A tryptic peptide containing these carboxymethylcysteine residues was isolated from digests of reduced and S-carboxymethylated lipoamide dehydrogenase and its amino acid sequence of 23 residues was determined. 3. The sequence is highly homologous with a similar sequence containing the active-site disulphide bridge of lipoamide dehydrogenase derived from the 2-oxoglutarate dehydrogenase complex of Escherichia coli (Crookes strain) and it is probable that, as in the bacterial enzyme, the disulphide bridge forms an intrachain loop containing six residues. The results indicate that the bacterial and mammalian proteins have a common genetic origin. 4. Amino acid sequences containing six other unique carboxymethylcysteine residues were also partly determined. 5. The analysis of the primary structure thus far is consistent with the view that the enzyme (mol.wt. approx. 110000) is composed of two identical polypeptide chains.

scholarly journals Derived amino acid sequence and identification of active site residues of Escherichia coli beta-hydroxydecanoyl thioester dehydrase.

1988 ◽  
Vol 263 (10) ◽  
pp. 4641-4646 ◽  
Author(s):  
J E Cronan ◽  
W B Li ◽  
R Coleman ◽  
M Narasimhan ◽  
D de Mendoza ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Active Site Residues

scholarly journals Amino acid sequence of the active site of Acanthamoeba myosin II.

1986 ◽  
Vol 261 (4) ◽  
pp. 1844-1848
Author(s):  
M A Atkinson ◽  
E A Robinson ◽  
E Appella ◽  
E D Korn
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Myosin Ii

scholarly journals The location of the active-site histidine residue in the primary sequence of papain

1968 ◽  
Vol 108 (5) ◽  
pp. 861-866 ◽  
Author(s):  
S. S. Husain ◽  
G. Lowe
Keyword(s):  
Amino Acid ◽  
Sodium Borohydride ◽  
Active Site ◽  
Iodoacetic Acid ◽  
Cysteine Residue ◽  
Histidine Residue ◽  
Primary Sequence ◽  
Active Site Cysteine ◽  
Active Site Histidine ◽  
Active Site Cysteine Residue

Papain that had been irreversibly inhibited with 1,3-dibromo[2−14C]acetone was reduced with sodium borohydride and carboxymethylated with iodoacetic acid. After digestion with trypsin and α-chymotrypsin the radioactive peptides were purified chromatographically. Their amino acid composition indicated that cysteine-25 and histidine-106 were cross-linked. Since cysteine-25 is known to be the active-site cysteine residue, histidine-106 must be the active-site histidine residue.

scholarly journals The complete amino acid sequence of chicken skeletal-muscle enolase

1986 ◽  
Vol 236 (1) ◽  
pp. 115-126 ◽  
Author(s):  
G A Russell ◽  
B Dunbar ◽  
L A Fothergill-Gilmore
Keyword(s):  
Skeletal Muscle ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Peptide Bond ◽  
Peptide Bonds ◽  
Complete Amino Acid Sequence ◽  
Arginine Residues ◽  
Cnbr Cleavage ◽  
Yeast Enolase ◽  
Chicken Skeletal Muscle

The complete amino acid sequence of chicken skeletal-muscle enolase, comprising 433 residues, was determined. The sequence was deduced by automated sequencing of hydroxylamine-cleavage, CNBr-cleavage, o-iodosobenzoic acid-cleavage, clostripain-digest and staphylococcal-proteinase-digest fragments. The presence of several acid-labile peptide bonds and the tenacious aggregation of most CNBr-cleavage fragments meant that a commonly used sequencing strategy involving initial CNBr cleavage was unproductive. Cleavage at the single Asn-Gly peptide bond with hydroxylamine proved to be particularly useful. Comparison of the sequence of chicken enolase with the two yeast enolase isoenzyme sequences shows that the enzyme is strongly conserved, with 60% of the residues identical. The histidine and arginine residues implicated as being important for the activity of yeast enolase are conserved in the chicken enzyme. Secondary-structure predictions are analysed in an accompanying paper [Sawyer, Fothergill-Gilmore & Russell (1986) Biochem. J. 236, 127-130].

Glutamine active site of formylglycinamide ribonucleotide amidotransferase. 2. Amino acid sequence of labeled peptides

Biochemistry ◽  
1977 ◽  
Vol 16 (6) ◽  
pp. 1070-1076 ◽  
Author(s):  
Shiro Ohnoki ◽  
Bor-Shyue Hong ◽  
John M. Buchanan
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site

scholarly journals Cloning and sequencing of protochlorophyllide reductase

1990 ◽  
Vol 265 (3) ◽  
pp. 789-798 ◽  
Author(s):  
P M Darrah ◽  
S A Kay ◽  
G R Teakle ◽  
W T Griffiths
Keyword(s):  
Amino Acid ◽  
Chemical Analysis ◽  
Amino Acid Sequence ◽  
Cdna Library ◽  
Avena Sativa ◽  
Cdna Clones ◽  
Lambda Phage ◽  
Cloning And Sequencing ◽  
Protochlorophyllide Reductase ◽  
Cnbr Cleavage

Putative protochlorophyllide reductase cDNA clones (252 and 113) were isolated from an etiolated-oat (Avena sativa) cDNA library. These were used to indirectly characterize a further clone, p127, isolated from a lambda-phage gt11 cDNA library. The latter (1.15 kb in length) was sequenced, and the derived amino acid sequence was shown to be remarkably similar to that derived from chemical analysis of a CNBr-cleavage fragment of the purified reductase, p127 codes for more than 95% of the reductase protein.

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