A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation

The Escherichia coli biotin holoenzyme synthetase, BirA, catalyzes transfer of biotin to the epsilon amino group of a specific lysine residue of the b iotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. Se quences of naturally biotinylated substrates are highly conserved across ev olutionary boundaries, and cross-species biotinylation has been demonstrate d in several systems. To define the minimal substrate requirements in BirA- catalyzed biotinylation, we have measured the kinetics of modification of a 23-residue peptide previously identified by combinatorial methods. Althoug h the sequence of the peptide bears Little resemblance to the biotinylated sequence in BCCP, it is enzymatically biotinylated in vivo. Rates of biotin transfer to the 23-residue peptide are similar to those determined for BCC P. To further elucidate the sequence requirements for biotinylation, transi ent kinetic measurements were performed on a series of amino- and carboxy-t erminal truncations of the 23-mer. The results, determined by stopped-flow fluorescence, allowed identification of a 14-residue peptide as the minimum required sequence. Additional support was obtained using matrix-assisted l aser desorption ionization time-of-flight (MALDI-TOF) mass spectrometric an alysis of peptides that had been incubated with an excess of biotinyl-5'-ad enylate intermediate and catalytic amounts of BirA. Results of these measur ements indicate that while kinetically inactive truncations showed no signi ficant shift in molecular mass to the values expected for biotinylated spec ies, kinetically active truncations exhibited 100% biotinylation. The speci ficity constant (k(cat)/K-m) governing BirA-catalyzed biotinylation of the 14-mer minimal substrate is similar to that determined for the natural subs trate, BCCP. We conclude that the 14-mer peptide efficiently mimics the bio tin acceptor function of the much larger protein domain normally recognized by BirA.