Abstract
The amino acid sequence of the enzyme cyanase (cyanate hydrolase) from Escherichia coli has been determined by automatic Edman degradation of the intact protein and of its component peptides. The primary peptides used in the sequencing were produced by cyanogen bromide cleavage at the methionine residues, yielding 4 peptides plus free homoserine from the NH2-terminal methionine, and by trypsin cleavage at the 7 arginine residues after acetylation of the lysines. Secondary peptides required for overlaps and COOH-terminal sequences were produced by chymotrypsin or clostripain cleavage of some of the larger peptides. The complete sequence of the cyanase subunit consists of 156 amino acid residues (Mr 16,350). Based on the observation that the cysteine-containing peptide is obtained as a disulfide-linked dimer, it is proposed that the covalent structure of cyanase is made up of two subunits linked by a disulfide bond between the single cystine residue in each subunit. The native enzyme (Mr 150,000) then appears to be a complex of four or five such subunit dimers.
Highlights
As in previous work (8, 9) we used strong acid for many of the peptide fractionations, and the identification of Asn and Gin could be ambiguous if the exposure to acid was sufficient to hydrolyze the amides
Frequent monitoring of the sequencer performance was carried out, and most of the reported data were obtained with repetitive yields from 93 to 97%
Tne general procedures used in this work., both those tnvo tvec in peptide sepa-eti on eno those in sequencing neve ceee described in detail (8,9)
Summary
As in previous work (8, 9) we used strong acid for many of the peptide fractionations, and the identification of Asn and Gin could be ambiguous if the exposure to acid was sufficient to hydrolyze the amides. 1. The amino acid sequence of E. coli cyanase.
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