Primary structure and enzymatic properties of carboxypeptidase II from wheat bran

Abstract

Wheat carboxypeptidase II has been isolated from wheat bran by affinity chromatography and its enzymatic properties and amino acid sequence has been determined. The enzyme is a dimer of molecular weight around 110,000 with each monomer composed of two peptide chains, an A-chain and a B-chain, linked by disulfide bridges. The A-chain exists in two forms, one, the A'-chain, being three amino acids shorter at the N-terminus, and consequently two different subunits, i.e. A-B and A'-B, and three different dimers, i.e. A'-B/A'-B, A'-B/A-B and A-B/A-B, are found. These different forms could be separated by ion exchange chromatography due to the A-and A'-chains differing by a single charge.Fragments of the A'-and B-chains, obtained by chemical cleavages with either cyanogen bromide or hydroxylamine and by enzymatic cleavages with either trypsin or S. aureus V8 protease, were sequenced and aligned to give the complete sequences of the two chains. The A'-and B-chains contain 260 and 160 amino acid residues, respectively. Three glycosylated asparagines are found in each chain. Alignment of the A-and B-chains with the corresponding chains of the known carboxypeptidases from germinated barley (malt) showed 95% homology with carboxypeptidase II and 37% homology with carboxypeptidase I. Similarly, the A-chain of wheat carboxypeptidase II exhibits 29% homology with the N-terminal part ofcarboxypeptidase Y and the B-chain exhibits 21% homology with the C-terminal part of carboxypeptidase Y.Like other serine carboxypeptidases wheat carboxypeptidase II exhibits peptidase activity with an acidic pH-optimum and esterase, amidase and peptidyl amino acid amide hydrolase activities with a basic pH optimum. The three different forms of the enzyme hydrolyse peptides with slightly different Km and k~at values but identical kcat/Km values. The specificity of the enzyme is identical to that of carboxypeptidase II from germinated barley: it exhibits a preference for basic and hydrophobic amino acid residues in the Pj and/or P/positions. Abbreviations: Bicine = N,N-bis(2-hydroxyethyl)glycine; BS = benzyl succinic acid; Bz = benzoyl; CABS-Sepharose = N-(e-aminocaproyl)-p-amino-benzylsuccinyl-Sepharose 4 B; Ches = 2(N-cyclohexylamino)ethane sulfonic acid; DPCC = diphenylcarbamyl chloride; EDTA = ethylenediamine tetraacetic acid, sodium salt; FA = furylacryloyl; Hepes = N-2-hydroxyethylpiperazine N'-2-ethanesulfonic acid; HPLC = high performance liquid chromatography; Mes = 2-(N-morpholino)ethanesulfonic acid; p-HMB = parahydroxy-mercuribenzoate; PTH = phenylthiohydantoin; TFA = trifluoroacetic acid. The binding site notation is that of SCHECHTER and BERGER (24).

An Expression and Bioinformatics Analysis of the Arabidopsis Serine Carboxypeptidase-Like Gene Family

“…These data suggest that many SCPL proteins may be posttranslationally modified as they are processed through the endoplasmic reticulum and Golgi. Finally, and as mentioned above, the inferred translations of many SCPL proteins contain a variable region known to be removed in CPDW-II (Breddam et al, 1987;Liao and Remington., 1990). Protein gel blots of SCT expressed in planta and in yeast suggest that this region is also removed during the maturation of this SCPL acyltransferase (Shirley and Chapple, 2003).…”

“…Second, two of these residues are situated on either side of a region that is highly variable among many SCPL family members. This variable region aligns with a sequence in CPDW-II that is similarly bordered by disulfide bond-forming Cys and is proteolytically excised from the mature enzyme (Breddam et al, 1987;Liao and Remington, 1990).…”

“…CPDW-II (Breddam et al, 1987) is 58% and 60% identical with respect to SCPL proteins 26 and 27. The hydroxynitrile lyase from Sorghum bicolor (accession no.…”

“…When these Arabidopsis SCPL proteins are aligned with carboxypeptidase II from wheat (Triticum aestivum; CPDW-II; GenBank accession no. A29639), a well-studied enzyme thought to be involved in the mobilization of protein reserves in seeds (Breddam et al, 1987), this variable region corresponds to a sequence that is proteolytically removed from the mature wheat protein (Breddam et al, 1987;Liao and Remington, 1990), suggesting that there may be few constraints on amino acid changes that occur within this region.…”

The primary structure of carboxypeptidase S3 from Penicillium janthinellum IBT 3991

“…carboxypeptidase Y [2,3], carboxypeptidase S 1 [4] and carboxypeptidase M3 [5] or enzymes containing two chains with approximately 260 and 160 amino acid residues, respectively (e.g. carboxypeptidases M1 [6] and M2 [7] and carboxypeptidase W2 [8]). These enzymes are synthesized as a single chain and then processed.…”