Human acylpeptide hydrolase. Studies on its thiol groups and mechanism of action.

The presence of a cysteine residue(s) near the active site of acylpeptide hydrolase was suggested by inactivation of the enzyme with sulfhydryl-modifying agents and by the substantial protection against inactivation afforded by the competitive inhibitor acetylmethionine. 5,5'-dithiobis-(2-nitrobenzoate) titrations of the native and the denatured enzyme together with analysis for cysteic acid after performic acid oxidation showed that the enzyme contained 12 free SH groups and three disulfide bonds/monomer. Chemical modification with radiolabeled iodoacetamide led to the labeling of Cys-30 and Cys-64 suggesting that one or both of these Cys residues are close to the active site. Modification of one or both of them probably inhibits the enzyme either because of a distortion of the active site or because the adducts present a barrier to the efficient diffusion of substrates into and products out of the active site. Studies on the mechanism of action of acylpeptide hydrolase have employed p-nitrophenyl-N-propyl carbamate as a potent active site-directed inhibitor. Enzyme inactivation, which follows pseudo first-order kinetics, is diminished by the competitive inhibitor acetylmethionine. The inhibited enzyme slowly regains activity at a rate that is increased in the presence of the nucleophile hydroxylamine. A general mechanism involving an acyl-enzyme intermediate is supported by evidence for the formation of acetyl-alanyl hydroxamate during hydrolysis of acetyl-alanine p-nitroanilide in the presence of hydroxylamine. The effect on Vmax and Km during this reaction indicate that hydrolysis of the acyl-enzyme intermediate is rate-limiting

Primary structure and tetrahydropteroylglutamate binding site of rabbit liver cytosolic 5,10-methenyltetrahydrofolate synthetase.

The primary sequence of 5,10-methenyltetrahydrofolate synthetase from rabbit liver was determined by amino acid sequencing of the purified enzyme. The enzyme contains 201 amino acid residues with a predicted mass of 22,779 Da. The enzyme is located in the cytosolic fraction of liver homogenates. Carbodiimide-activated 5-formyltetrahydropteroylmonoglutamate and the pentaglutamate form of the substrate both irreversibly inactivate the enzyme by forming a covalent bond to Lys-18. Non-activated 5-formyltetrahydropteroylpentaglutamate protected against this inactivation. Substrate specificity studies showed that increasing the number of glutamate residues from zero to five on 5-formyltetrahydropteroate results in a 2 order of magnitude increase in the affinity of the substrate for the enzyme but only a 3-fold increase in the value of Vmax

Primary structure of a protease isoinhibitor from bovine spleen. A possible intermediate in the processing of the primary gene product.

Sequence studies on the protease isoinhibitor I isolated from bovine spleen have revealed that it consists of two molecular variants which differ only in the presence of an additional COOH-terminal residue, asparagine, in the less abundant form. The complete amino acid sequence shows that they are composed of 65 or 66 residues and predicts Mr of 7223 or 7338, respectively. The sequences correspond exactly to the 58-residue polypeptide chain of spleen isoinhibitor II plus NH2- and COOH-terminal extensions of 2 and 5 or 6 amino acid residues, respectively. Moreover the entire sequences are located within the 100-residue structure deduced from the mRNA and DNA sequences of the putative precursor. These data support the idea that the molecular variants of isoinhibitor I are either mature proteins with distinct functional roles, or intermediates in the multistage processing of the primary product of gene expression, which eventually leads to the mature protein, i.e. inhibitor II

The primary structure of mitochondrial aspartate aminotrasferase from human heart

The complete amino acid sequence of the mitochondrial asparate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) from human heart has been determined based mainly on analysis of peptides obtained by digestion with trypsin and by chemical cleavage with cyanogen bromide. Comparison of the sequence with those of the isotopic isoenzymes from pig, rat and chicken showed 27, 29 and 55 differences, respectively, out of a total of 401 amino acid residues. Evidence for structural microheterogeneity at position 317 has also been obtained

The primary structure of rabbit liver mitochondrial serine hydroxymethyltransferase.

The complete amino acid sequence of mitochondrial serine hydroxymethyltransferase from rabbit liver was determined. The sequence was obtained from analysis of peptides isolated from chymotryptic, cyanogen bromide, and limited acid cleavages of the protein. The enzyme consists of four identical subunits, each of 475 residues, i.e. 8 residues shorter than the subunit of the corresponding cytosolic isoenzyme. The sequences of the two rabbit proteins are easily aligned, provided a gap of 5 residues near the amino terminus and a gap of 3 residues near the carboxyl terminus are included in the mitochondrial sequence. The overall degree of identity between the two isoenzymes is 61.9%, whereas the structural identity of each eukaryotic isoenzyme with the corresponding Escherichia coli enzyme is about 40%. The rabbit isoenzymes are about 70 residues longer than the E. coli enzyme, with one-half of these residues accounted for by insertions in both isoenzymes near their carboxyl terminus. Predictions of secondary structure and calculations of hydropathy profiles are also presented, suggesting an even more extensive degree of identity in the three-dimensional folding of the three proteins, in accord with the known similarity of their catalytic properties. Evidence was obtained for the existence of additional molecular forms of the mitochondrial protein, differing in the absence of some amino acid residues at the amino terminus of the polypeptide chain

A tetrameric iron superoxide dismutase from the eucaryote Tetrahymena pyriformis.

Abstract An iron-containing superoxide dismutase has been purified from the protozoan Tetrahymena pyriformis. It has a molecular weight of 85,000 and is composed of four subunits of equal size. The tetramer contains 2.5 g atoms of ferric iron. Visible absorption and electron spin resonance spectra closely resemble those of other iron-containing superoxide dismutases. The amino acid sequence of the iron superoxide dismutase was determined. Each subunit is made up of 196 residues, corresponding to a molecular weight of 22,711. Comparison of the primary structure with the known sequences of other iron-containing superoxide dismutases reveals a relatively low degree of identity (33-34%). However, a higher percentage identity is found with mammalian manganese-containing superoxide dismutases (41-42%). The amino acid sequence is discussed in consideration of residues that may distinguish iron from manganese or dimeric from tetrameric superoxide dismutases

Nitrite reductase from Pseudomonas aeruginosa: Sequence of the gene and the protein

AbstractThe gene coding for nitrite reductase of Pseudomonas aeruginosa has been cloned and its sequence determined. The coding region is 1707 bp long and contains information for a polypeptide chain of 568 amino acids. The sequence of the mature protein has been confirmed independently by extensive amino acid sequencing. The amino-terminus of the mature protein is located at Lys-26; the preceding 25 residue long extension shows the features typical of signal peptides. Therefore the enzyme is probably secreted into the periplasmic space. The mature protein is made of 543 amino acid residues and has a molecular mass of 60204 Da. The c-heme-binding domain, which contains the only two Cys of the molecule, is located at the amino-terminal region. Analysis of the protein sequence in terms of hydrophobicity profile gives results consistent with the fact that the enzyme is fully water soluble and not membrane bound; the most hydrophilic region appears to correspond to the c-heme domain. Secondary structure predictions are in general agreement with previous analysis of circular dichroic data