J. Biol. Chem., Vol. 256, Issue 20, 10293-10305, Oct, 1981
Primary structure of 3-phosphoglycerate kinase from horse muscle. II. Amino acid sequence of cyanogen bromide peptides CB1-CB4 and CB6-CB14, sequence of methionine-containing regions, and complete sequence of the enzyme
M Merrett
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.
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