The crystal structure of carboxypeptidase T from Thermoactirzomyces vulgaris has been determined at 0.235-nm resolution by X-ray diffraction. Carboxypeptidase T is a remote homologue of mammalian Zn-carboxypeptidases. In spite of the low degree of amino acid sequence identity, the threedimensional structure of carboxypeptidase T is very similar to that of pancreatic carboxypeptidases A and B. The core of the protein molecule is formed by an eight-stranded mixed p sheet. The active site is located at the C-edge of the central (parallel) part of the B sheet. The structural organization of the active centre appears to be essentially the same in the three carboxypeptidases. Amino acid residues directly involved in catalysis and binding of the C-terminal carboxyl of a substrate are strictly conserved. This suggests that the catalytic mechanism proposed for the pancreatic enzymes is applicable to carboxypeptidase T and to the whole family of Zn-carboxypeptidases. Comparison of the amino acid replacements at the primary specificity pocket of carboxypeptidases A, B and T provides an explanation of the unusual 'A+B' type of specificity of carboxypeptidase T. Four calcium-binding sites localized in the crystal structure of carboxypeptidase T could account for the high thermostability of the protein.Metallocarboxypeptidases are exopeptidases which contain a zinc ion in the active centre and catalyze the hydrolysis of C-terminal amino acids from polypeptide substrates. Extensive studies of bovine pancreatic carboxypeptidase A (CPA) by different techniques including crystallography, spectroscopy, kinetics and site-directed mutagenesis resulted in the understanding of the general principles of the catalytic mechanism (Christianson and Lipscomb, 1989). Three-dimensional structures have been determined for pancreatic carboxypeptidases A (Rees et al., 1983;Kobe and Goldsmith, 1990) and B (Schmid and Herriott, 1976; Coll et al., 1991) which share a common polypeptide fold but differ in substrate specificity (Hartsuck and Lipscomb, 1971 ;Folk, 1971 polypeptide chain of 326 amino acids and contains one zinc ion (Osterman et al., 1984). The amino acid sequence of CPT, deduced from the gene encoding the protein (Smulevitch et al., 1991), shows only moderate similarity with the CPA and CPB sequences; 30% and 27% identical residues, respectively. CPT exhibits an unusual 'dual'-substrate specificity combining features of both CPA and CPB; it is able to split off hydrophobic and basic amino acids with comparable efficiency (Osterman et al., 1984). As other proteins from Thermoactinomyces, CPT has an increased thermal stability in the presence of calcium ions (Osterman et al., 1984).Structure/functional studies of CPT provide a possibility to study the general principles of catalysis by Zn-carboxypeptidases. On the basis of the comparative primary-structure analysis, all Zn-carboxypeptidases were divided into two groups ; ,,digestive,, and ,,regulatory,, enzymes (Osterman et al., 1992). Pancreatic CPA and CPB appear to be structurally ...

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A serine proteinase was isolated from fruits of Maclura pomifera (Raf.) Schneid. by affinity chromatography on bacitracin-containing sorbents and gel-filtration. The enzyme, named macluralisin, is a glycoprotein with a molecular mass of 65 kDa; its protein moiety corresponds to a molecular mass of 50 kDa. The substrate specificity of macluralisin towards synthetic peptides and insulin B-chain is similar to that of cucumisin, a subtilisin-like proteinase from melon fruit. The enzyme is completely inhibited by diisopropylfluorophosphate. Its amino-acid composition resembles that of a serine proteinase isolated from the Cucurbitaceae. The N-terminal sequence has 33% of its residues identical to those of the sequence of fungal subtilisin-like proteinase K. Hence, Maclura pomifera serine proteinase belongs to the subtilisin family, which seems to be broadly distributed in the plant kingdom.

Latex of dandelion roots contains a serine proteinase that hydrolyzes a chromogenic peptide substrate Glp-Ala-AlaLeu-pNA optimally at pH 8.0. Maximal activity of the proteinase in the roots is attained in April, at the beginning of plant development after the winter period. The protease was isolated by ammonium sulfate precipitation of the root extract followed by affinity chromatography on a Sepharose-Ala-AlaLeu-mrp and gel filtration on Superose 6R performed in FPLC regime. Pure serine proteinase named taraxalisin was inactivated by specific inhibitors of serine proteinases, diisopropylfluorophosphate (DFP) and phenylmethylsulfonylfluoride (PMSF). Its molecular mass is 67 kDa and pI 4.5. pH stability range is 6^9 in the presence of 2 mM Ca P+ , temperature optimum is at 40³C; K m =0.37 þ 0.06 mM. The substrate specificity of taraxalisin towards synthetic peptides and insulin B-chain is comparable with that of two other subtilisin-like serine proteinases, cucumisin and macluralisin. The taraxalisin N-terminal sequence traced for 15 residues revealed 40% coinciding residues when aligned with that of subtilisin Carlsberg.z 1998 Federation of European Biochemical Societies.