Amino Acid Sequence of Escherichia coZi Biotin Carboxyl Carrier Protein (9100)*

The amino acid sequence of a proteolytic fragment of Escherichin coli hiotin carboxyl carrier protein was determined from the structures of overlapping tryptic, thermoly- tic, and staphylococcal protease peptides together with automated sequenator analyses on the intact protein. The fragment, 82 residues in length, contains the single residue of biocytin of’the protein. The relationship of the .M,. = 9100 fragment to the native .M,. = 22,500 subunit is discussed. Biotin carboxyl carrier protein plays a central role in the acetyl-CoA carboxylase complex of Escherichin coli. The enzyme complex catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, the first committed step in the synthesis of long chain fatty acids. The enzyme has been resolved into three dissimilar subunits (1, 2). In addition to BCCP,’ there is a biotin carboxylase and a transcarboxylase, each catalyzing one step in the overall reaction:

Electrophoretic mobilities at pH 6.5 were determined by high voltage paper electrophoresis.
Four additional peptides, all arising as a result of chymotryptic-like cleavages, were obtained. These were T4, T5, T6, and T8. The first three were completely sequenced by the dansyl procedure. T5 contained the single cysteinyl residue and T6 the single biocytinyl residue of the protein. As the result of limited amounts of material, only the NH,-terminal residue of T8 was determined. Peptides corresponding to residues 20 to 26, 48 to 57, and 52 to 57 could be tentatively identified in impure fractions, but were not obtained in a homogeneous form.
The thermolytic digest of BCCP,,,,,, produced negligible acidinsoluble material, and hence yielded an almost complete set of peptides. For the five regions (residues 35 to 36,43 to 46,49, 61 to 63, and 81 to 82) for which no peptides are listed (Fig. l), impure fractions were again obtained in which the missing peptides could be tentatively identified, albeit in low yield. Amino acid and NH,-terminal analyses of Th5 showed it to be heterogeneous, with partial phenylalanine at the NH, terminus. No attempt was made to further purify the components of this pool.
The S. aureus protease hydrolysis of S-[Wlcarboxymethyl BCCP,, produced a complete set of peptides in generally high yield. The peptides were fractionated on Sephadex G-50 followed by chromatography on ion exchange columns as described in the miniprint supplement and were structured by the dansyl procedure.
Amide Assignments -The amide assignments of all of the aspartyl and glutamyl residues of the protein are listed in Table II. Glu 3 and Asp 24 were assigned as acids from the sequenator analyses. Glu 30 was assigned as an acid based on carboxypeptidase A hydrolysis of SP2b. Position 33 was assigned as glutamine from digestion of T3a with carboxypeptidases A and B. Asn 36 and Asn 39 were identified from the electrophoretic mobilities of T4 and Th7. Residue 45 was identified as glutamic acid from carboxypeptidase A hydrolysis of SP3 and residues 51 and 52 were identified as amides from the neutral electrophoretic behavior of ThlO at pH 6.5. Carboxypeptidase A hydrolysis of SP4 released 1 mol of glutamic acid per mol of peptide. Since residue 52 is glutamine, it follows that residue 54 is glutamic acid. Asp 56 was identified from the thiohydantoin.
Residues 67 and 73 were identified as glutamic acid by carboxypeptidase A hydrolysis of SP5 and SP6, respectively. Gln 70, Asp 75, and Glu 76 were identified from the thiohydantoins.
The COOH terminus of the protein was identified as glutamic acid by carboxypeptidase A digestion of BCCP,, . quenator analysis provides identification of the first 24 residues. Tryptic, thermolytic, and Staphylococcus aureus protease peptides derived from this region are consistent with the structure with respect to both composition and to residues positioned by sequence analysis. The extension of this region to peptide SP2b is provided mainly by the compositional overlap of SP2. The structure of SP2a has been partially determined by subdigestion with trypsin and is readily added to the completely structured SP2b to yield SP2. SP2b overlaps both T3 and T3a as well as Th6 and ThGa, which extends the sequence to Lys 34. These same peptides are in turn overlapped by SP3 which extends the sequence to Asn 39 in directly structured residues and by composition to Glu 45. This provides the information to overlap peptides T4 and T5, which are both fully sequenced. The compositional overlap provided by the COOH-terminal portion of SP3 is secured by the fact that it contains the only cysteinyl residue in the protein. The Ala-Met sequence in the COOH-terminal position of T5 is overlapped by SP4, which extends the sequence through the biocy-tiny1 residue at position 48 to Glu 54. The overlap of SP4 and SP5 is provided by Thll. This 2-residue overlap of Ile-Glu deserves further comment. Two other Ile-Glu sequences occur in the protein, at positions 29-30 and 81-82. However, the sequence at 29-30 is rigorously extended by peptides T3 and Th6 and therefore cannot be confused with the Ile-Glu at position 53-54. The sequence at 81-82, which forms the COOH terminus of the protein, is contiguously extended from position 53-54, as described below, thus making an overlap impossible. SP5, which was completely structured, extends the sequence to residue 67. Th12 provides a firm overlap to SP6. By composi-