Primary Structure of Tyrosinase from Neurospora crassa I. PURIFICATION AND AMINO ACID SEQUENCE OF THE CYANOGEN BROMIDE FRAGMENTS*

Cyanogen bromide (CB) cleavage of Neurospora ty- rosinase resulted in four major fragments, CB1 (222 residues), CB2 (82 residues), CB3 (68 residues), and CB4 (35 residues), and one minor overlap peptide CB2-4 (117 residues) due to incomplete cleavage of a methionyl- threonyl bond. The sum of the amino acid residues of the four major fragments matches the total number of amino acid residues of the native protein. The amino acid sequences of the cyanogen bromide fragments CB2, CB3, and CB4 were determined by a combination of automated and manual sequence analysis on pep- tides derived by chemical and enzymatic cleavage of the intact and the maleylated derivatives. The peptides were the products of cleavage by mild acid hydrolysis, trypsin, pepsin, chymotrypsin, thermolysin, and Staphylococcus aureus protease V8. The cyanogen bromide fragment CB1 was found to contain two unusual amino acids whose chemical structure will be presented in the following paper.

Cyanogen bromide (CB) cleavage of Neurospora tyrosinase resulted in four major fragments, CB1 (222 residues), CB2 (82 residues), CB3 (68 residues), and CB4 (35 residues), and one minor overlap peptide CB2-4 (117 residues) due to incomplete cleavage of a methionylthreonyl bond. The sum of the amino acid residues of the four major fragments matches the total number of amino acid residues of the native protein. The amino acid sequences of the cyanogen bromide fragments CB2, CB3, and CB4 were determined by a combination of automated and manual sequence analysis on peptides derived by chemical and enzymatic cleavage of the intact and the maleylated derivatives. The peptides were the products of cleavage by mild acid hydrolysis, trypsin, pepsin, chymotrypsin, thermolysin, and Staphylococcus aureus protease V8. The cyanogen bromide fragment CB1 was found to contain two unusual amino acids whose chemical structure will be presented in the following paper.
The enzyme occurs in different microorganisms, plants and animals and is mainly involved in the biosynthesis of melanins and other polyphenolic compounds (2)(3)(4). Tyrosinases from the common mushroom Agaricus bisporus and Neurospora crassa contain an antiferromagnetically spin-coupled copper pair (5)(6)(7)(8) which in the cuprous state binds molecular oxygen reversibly (9,10). Resonance Raman studies have shown that the oxygen in Neurospora oxytyrosinase is bound to the protein as peroxide ( I l ) , as was demonstrated earlier for oxyhemocyanin (12). In contrast to the wealth of information available on the mode of copper binding at the active site (8,13) of tyrosinase, the knowledge of the covalent structure of the polypeptide chains is still very limited due to the occurrence of tnolecular heterogeneity (2,4).
Tyrosinase from the ascomycete N. crassa has been reported to be a monomeric protein with a molecular weight of 33,000 (14,15). More recently, this enzyme was found to consist of a single polypeptide chain of M , of about 44,000 (10). To arrive a t a better understanding of the structure and * This work has been supported by Schweizerischer Nationalfonds Grants 3.018-0.76 and 3.420-0.78. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Sect.ion 1734 solely to indicate this fact.
$ To whom inquiries should be addressed.
function of this copper-containing monooxygenase, we have carried out the determination of the complete amino acid sequence of Neurospora tyrosinase. In the first paper, the isolation and the sequence analysis of the cyanogen bromide fragments CB1' to CB4 are described.
The accompanying paper reports the complete primary structure of Neurospora tyrosinase (16). A preliminary report of some of this work has been published (17).

RESULTS AND DISCUSSION
Purification of Cyanogen Bromide Peptides-Neurospora tyrosinase is a single-chain protein with a molecular weight of 46,000 (10,17). In order to determine its amino acid sequence, the protein was cleaved with cyanogen bromide in 75% trifluoroacetic acid. As predicted from the presence of three methionine residues, four major fragments (CBl, CB2, CB3, and CB4) were obtained. The peptides were isolated by gel filtration on Sephadex G-100 (Fig. 1). The fragments CB1 and CB4 were found to be sufficiently pure for sequence analysis. The mixture of CB2 and CB3 was resolved by repeated gel chromatography on Sephadex G-50 (superfine) in 7% formic acid (Fig. 3). Alternatively, the mixture was maleylated and subsequently separated by DEAE-cellulose chromatography (

Amino Acid
Sequence of Neurospora Tyrosinase 6409 scribed, an overlap peptide of CB2 and CB4 which arises from incomplete cleavage of a methionyl-threonyl bond in this solvent (Fig. 2). This fragment was obtained in pure form by rechromatography on Sephadex G-50 (superfine) in 7% formic acid (Fig. 4). Characterization of Cyanogen Bromide Peptides-Amino acid compositions, molecular weights, NH? termini, and yields of the cyanogen bromide fragments are listed in Table I. The amino acid compositions obtained from amino acid analysis are in good agreement with those determined by sequence analysis (shown in parentheses). The sum of the four major fragments CB1 to CB4 corresponds closely to the composition of native Neurospora tyrosinase (last column). The amino acid composition of the minor overlap peptide CB2-4 is in good agreement with the sum of the compositions of CB2 and CB4. Thus, all amino acids of Neurospora tyrosinase are contained within the four cyanogen bromide fragments CB1 to CB4.
Amino Acid Sequence of Fragment CBl-The elucidation of the amino acid sequence of the cyanogen bromide fragment CB1 relies to a great extent on the tryptic peptides of the unmodified fragment and of the maleylated derivative (Fig.  6A). With the exception of two aggregated peptides designated as Tc (core peptides) all tryptic peptides were isolated in good yields. These peptides yielded more than 89% of the total primary structure of CB1 including the sequence information of residues 1 to 35 which was obtained by automated sequence analysis of the unmodified fragment. For details, see Miniprint. For the alignment of the tryptic peptides of CBl, suitable overlap peptides were obtained by digestion of the maleylated derivative with Staphylococcus aureus protease and b y cleavage of the unmodified fragment with dilute acetic acid (Fig. 6A). To complete the NH2-terminal sequence of CBl and to unambiguously align the remaining tryptic peptides, the unmodified fragment was further cleaved with pepsin, chymotrypsin, and thermolysin (Fig. 6 B ) . For details of the isolation and sequence analyses of these peptides, see Miniprint.
CB1 is the largest of the four cyanogen bromide fragments of Neurospora tyrosinase, containing 222 amino acid residues. As a consequence of its size, a large number of peptides (>140) had to be isolated to arrive at the primary structure. Despite the efforts made in the course of this work in obtaining large fragments of CB1, the major portion of the amino acid sequence was eventually derived from small peptides generated by different proteases.
Amino Acid Sequence of Fragment CB2-The amino acid sequence of the cyanogen bromide fragment CB2 was determined by a combination of automated and manual sequence analysis of the intact fragment and of peptides derived from proteolytic cleavage of CB2 and its maleylated derivative with trypsin, thermolysin, and pepsin (Fig. 7). Complete documentation of the isolation and sequence analyses of these peptides is given in the Miniprint.
The cyanogen bromide fragment CB2 contains 82 of the 407 amino acid residues of Neurospora tyrosinase. It is devoid of histidine and homoserine; hence, it is placed at the COOHterminal end of the intact protein.
Amino Acid Sequence of Fragment CB3-Like native Neurospora tyrosinase, the cyanogen bromide fragment CB3 was found to be resistant to Edman degradation. Chromatography of the tryptic digest of CB3 on the ion exchange resin "72 gave the pentapeptide T3 (Fig. 8), which was blocked. The amino acid sequence of this peptide is N-acetyl-Ser-Thr-Asp-Ile-Lys, previously determined by computer-assisted mass spectrometry (32). The complete amino acid sequence of CB3 (Fig. 8) was determined by automatic and manual sequence analysis on peptides generated by cleavage with trypsin, dilute acetic acid, and thermolysin. For details, see Miniprint.
The cyanogen bromide fragment CB3 is composed of 68 residues and is devoid of tryptophan. It contains an Asn-Gly bond (residues 18 and 19) whose presence was confirmed by the isolation of two fragments after hydroxylamine cleavage (29) of peptide T h l (Fig. 8). Finally, the amide assigcments of the aspartic acid residues of CB3 were found to be in complete agreement with the known specificity of cleavage at Asp by dilute acetic acid (33). Amino Acid Sequence of Fragment C34"The primary structure of CB4 was determined by a combination of automated and manual sequence analysis of peptides derived from proteolytic cleavage of the unmodified fragment with thermolysin, pepsin, and chymotrypsin (Fig. 9). For details of isolation and sequence analyses of these peptides, see Miniprint.
The cyanogen bromide fragment CB4 consists of 35 amino acid residues (Fig. 9) lacking lysine and tyrosine. It shows a high content of hydrophobic amino acids (438) including 3 out of a total of 12 tryptophans in Neurospora tyrosinase. This unusual amino acid composition could at least partially be responsible for the observed resistance of cleavage of the Arg 21-Leu 22 bond by trypsin.