Primary structure of streptococcal proteinase. III. Isolation of cyanogen bromide peptides: complete covalent structure of the polypeptide chain.

The following sequence has been derived for streptococcal proteinase. (See article). The sequence permits the assignment of the single cysteine residue essential for catalytic action at position 47 from the NH2 terminus of the protein. The tryptophan residue at the binding site of the enzyme is at position 214. A histidine residue at position 195 has been assigned as the catalytically important entity in the molecule. Streptococcal proteinase and papain, an enzyme with similar properties, are compared with respect to structure and function.

The preceding communications (1,2) (N,-saturated) by warming the suspension to 37" for 30 min, and cyanogen bromide (1 g) was added. After 24 hours at room temperature, the reagents were removed under a stream of Nz. The residue was taken up in 5 ml of 20% acetic acid and applied to a column of Sephadex G-50 equilibrated with the same solvent (Fig. 1A).
SEPHADEX G-50 (300x2.5cm) _ Cleavage by cyanogen bromide was also performed on the protein at 37" for 12 hours under nitrogen in the presence of tryptamine (200 mg/200 mg of protein (4). The product was fractionated on Sephadex G-50 as described for Fig. 1A The combined peptides (0.23 cmol) were dissolved in 8 M urea buffered in 0.1 h Tris-HCI, pH 8.6. Dithiothreitol (7.7 mg) was added, and the mixture was incubated at 30" under N, for 24 hours. The peptide solution was adjusted to 50% acetic acid and desalted on a Sephadex G-25 column in the same solvent (Fig. 5). Based on the recovery of radioactivity, 86% of the alkylated methionines were regenerated as methionine. The amino acid composition of this regenerated peptide, II-R (Table IV) showed 2.2 residues of methionine.
The expected number of methionines for this peptide is 3.    (4); serine and threonine were corrected by extrapolation.
The 72.hour values were used for valine and isoleucine. The integral numbers are the results of the determination of the sequence (Fig. 2)

DISCUSSION
The complete amino acid sequence of streptococcal proteinase is shown in Fig. 2. Under each amino acid are the helical (a) potentials in the first row and fl structure potentials (8) in the second row, based on protein conformation prediction rules derived by Chou and Fasman (11,12). The conformational parameters Pru and P/3 for amino acids in helices and @ structures, respectively, were utilized to predict helical regions and p structures in the proteinase. Inspection of the models (Fig. 2) (7) 7.01 (7) 6.54 (7)  7 6.20 (7) Glutamlc Acldr 4.36 (4)  showed no significant repeating sequences in the molecule. The uneven distribution of residues over the chain is marked; tryptophan, phenylalanine, glycine, and arginine are predominantly in the COOH-terminal half of the protein, whereas 9 out of 10 threonine residues are in the NH*-terminal half. The sequence in Fig. 2 permits the location of residues in proteinase that have special reactivities.
The catalytically essential sulfhydryl group (10) is located at position 47, which is at the beginning of p sheet region [47][48][49][50][51][52][53][54][55][56] showed that treatment of streptococcal proteinase with 2hydroxy-5-nitrobenzyl bromide resulted in the modification of a single tryptophan residue and a concomitant loss of enzymic activity. From a tryptic hydrolysate, he isolated a peptide which by reference to Fig. 2 could arise only from CB5-T(m)-5 with a chymotryptic-like cleavage at an aromatic residue at position 208 (2). Thus, the tryptophan residue believed to be at the binding site of the enzyme is Trp,,,.
Liu showed (14) that 1 histidine residue is specially reactive toward o-N-bromoacetyl-L-arginine methyl ester and that the alkylated protein is inactive. Tai and Liu' have assigned Hislss at the boundary of the helix-coil region 195-202 to be the catalytically essential histidine residue in the proteinase. The propensity for polar active site residues at the helix-coil boundaries of enzymes has been attributed (11) to the fact that these regions are more flexible than the rigid inner helix core, thus facilitating substrate binding as well as enzyme catalysis.
Streptococcal proteinase is a sulfhydryl protease similar to papain. The two enzymes have similarities as well as differences (8, 15). Similarities include an active site with a highly reactive -SH group (14,16,17) and an imidazole ring (14,16,17,18), similar specificity toward the phenylalanine-chain of insulin (19,20), and activity in 8 M urea (7,21). The differences reside in size (253 amino acid residues for proteinase and 212 residues for papain), in the absence of S-S cross-links in the streptococcal proteinase, and in the absence of any special similarities in the amino acid compositions of the two proteins. The proteinase exhibits a high ratio of esterase to peptidase activity (22), whereas papain hydrolyzes both types of substrates at about the same rate (23).
The primary structural studies of the two enzymes revealed the following similarities.
The highly reactive -SH group in both enzymes is located in the NH*-terminal portion of the protein (papain, Cys,,; proteinase Cys,,), while the tryptophan residue believed to be at the binding site of the enzyme is positioned at the COOH-terminal portion of the molecule (papain, Trp,,,; proteinase, Trpzlr). The catalytically crucial histidine residue in papain (HisIs@) and in the proteinase (His& is at about the same distance (18 or 19 amino acid residues) from the crucial tryptophan residue in each protein. Moreover, the sequence around the reactive cysteine of both enzymes is Gly-X-Cys (Fig. 6) as is the case for all known thiol proteinases (24). In both enzymes the reactive tryptophan residue is next to a glycine residue (Fig. 7), and the catalytically important histidine residue is next to an alanine residue * Unpublished observation. Sequence similarities between papain and streptococcal proteinase near the active center. (Fig. 7 and Ref. 18). Limited homologies found between papain and the proteinase could represent converging evolution of active sites that have similar specificities or they could be the vestiges of a primitive active site conserved during diverging evolution of the two proteins.