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Acetyl coenzyme A carboxylase. Proteolytic modification of biotin carboxyl carrier protein.

Abstract The biotin carboxyl carrier protein (BCCP) component of Escherichia coli acetyl coenzyme A carboxylase has been previously isolated in multiple active forms, ranging in molecular weight from 45,000 to 9,100. Since the apparent native form of BCCP is the largest of these species, the isolation of the small forms was attributed to proteolysis during the purification procedures employed. We have now shown that native BCCP is very susceptible to subtilisin hydrolysis. Limited proteolysis of crude or purified preparations of native BCCP with subtilisin Carlsberg produces a mixture of BCCP species analogous to those previously noted. After more extensive proteolysis with subtilisin Carlsberg, all the intermediate forms are converted to a small form of BCCP, termed BCCP(sc), which we have isolated in milligram quantities and compared with BCCP(9100), the smallest form of BCCP previously isolated (mol wt 9100). Purified BCCP(sc) was found to be homogeneous by electrophoresis in polyacrylamide gels in the presence or absence of sodium dodecyl sulfate, and in both gel systems it was indistinguishable from BCCP(9100), indicating the similarity in size and charge between the two proteins. From isoelectric focusing in polyacrylamide gels, an isoelectric point of approximately 4.5 was determined for both BCCP(sc) and BCCP(9100). The amino acid composition of BCCP(sc) (80 amino acid residues, including 1 half-cystine residue) was identical with that of BCCP(9100), except that BCCP(9100) contains 2 additional alanine residues. The single sulfhydryl residue in either protein was titratable with 5,5'-dithiobis(2-nitrobenzoic acid) only when denaturants such as 8 m urea or 1% sodium dodecyl sulfate were added. The biotin content of both proteins is consistent with the presence of 1 biotin residue per molecule. BCCP(sc) was active as a carboxyl acceptor and donor in the biotin carboxylase and transcarboxylase reactions of acetyl-CoA carboxylase. In each of these reactions a Km of approximately 3 x 10-5 m was determined for BCCP(sc), compared with a value of 2.5 x 10-5 m for BCCP(9100). Vmax values for the two proteins in these reactions were essentially identical. When BCCP(10400) (96 amino acid residues; mol wt 10400), previously isolated along with BCCP(9100) from a mixture of small BCCP species, was treated with subtilisin Carlsberg, it was quantitatively hydrolyzed to a species of BCCP identical with BCCP(sc). BCCP(sc) and BCCP(9100) are resistant to further proteolysis at 37° and pH 8 by high concentrations of subtilisin as well as trypsin, chymotrypsin, and thermolysin, lending support to the idea that these small forms represent a stable "core" peptide in native BCCP. Efforts to isolate the peptide fragment lacking biotin from subtilisin-treated BCCP have been unsuccessful, suggesting that it is rapidly degraded by subtilisin.