Abstract
The amino acid sequences of 13 of the 14 cyanogen bromide peptides of horse muscle 3-phosphoglycerate kinase have been determined. These peptides together constitute 75% of the structure of the enzyme. Except for the smallest peptides, automated sequence analysis of the parent peptide was employed together with the automated or manual (5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Edman method) sequencing of relevant peptides obtained by proteolytic digestion. In the case of the five smallest peptides, all between 6 and 11 residues in length, sequences were derived from analyses of the intact peptides. CB14a, a peptide produced in low yield during the preparation of the cyanogen bromide fragments, was helpful in the sequence analysis of CB14. This peptide appeared to have arisen as the result of a cyanogen bromide cleavage on the carboxyl side of a tryptophanyl residue. The sequences of these 13 cyanogen bromide peptides, together with that of CB1, reported previously (Hardy, G. W., Darbre, A., and Merrett, M. (1981) J. Biol. Chem. 256, 10284-10292), have been combined with data obtained from the sequence analysis of tryptic peptides derived from the methionine-containing regions of the enzyme to determine the complete sequence. Isolation of these overlap peptides was facilitated by the labeling of methionyl residues with iodo[2-14C]acetic acid prior to tryptic digestion. Tryptophan-containing tryptic peptides were also isolated, and their sequence analysis enabled the positions of the four tryptophanyl residues to be established. In this way, the problem caused by tryptophan degradation during the preparation of the cyanogen bromide fragments was overcome. The NH2 terminus of CB1, the NH2-terminal cyanogen bromide peptide, was identified as N-acetyl serine by mass spectrometry. The alignment of the cyanogen bromide fragments of horse muscle 3-phosphoglycerate kinase described here is in complete agreement with that based on X-ray analysis of the enzyme reported previously (Banks, R. D., Blake, C. C. F., Evans, P. R., Haser, R., Rice, D. W., Hardy, G. W., Merrett, M., and Phillips, A. W. (1979) Nature 279, 773-777). The complete structure is comprised of 416 residues and has a molecular weight of 44,519.
Highlights
The amino acid sequences of 13 of the 14 cyanogen bromide peptides of horse muscle 3-phosphoglycerate kinase have been determined
Except for the smallest peptides, automated sequence analysis of the parent peptide was employed together with the automated or mgnual(5dimethylaminonaphthalene-1-sulfonyl-Edman method) sequencing of relevant peptides obtained by proteolytic digestion
CB14a, a peptide produced in low yield during the preparation of the cyanogen bromide fragments, was helpful in the sequence analysis of CB14
Summary
CBl-CB4 and CB6-CB14 are described in the miniprint supplement. Alignment of the CNBr Fragments in the Complete Sequence-An unambiguous alignment of the CNBr peptides of phosphoglycerate kinase has been ascertained in the following manner. The blocked NH,-terminal residue of CBI and the absence of homoserine from CB14 enabled these two peptides to be located at the NHz-terminal and COOH-terminal ends of the molecule, respectively. 2-4 and 6-9, Tables I-XLI, and additional references) are presented in miniprint at the end of this paper. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No 81M-1394, cite author, and include a check for $27.00 per set of photocopies. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press. ‘The abbreviations used are: CNBr, cyanogen bromide; DMAA, N-dimethylallylamine; Quadrol, N,N,N’,N’.-tetrakis(2-hvdroxvnropyl)ethylenediamine; PTH, phenylthiohydantoin; Cm, S-carboxymethyl; TLC, thin layer chromatography; dansyl, J-dimethylaminonaphthalene-l-sulfonyl
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