Abstract
Biotin protein ligases catalyze specific covalent linkage of the coenzyme biotin to biotin-dependent carboxylases. The reaction proceeds in two steps, including synthesis of an adenylated intermediate followed by biotin transfer to the carboxylase substrate. In this work specificity in the transfer reaction was investigated using single turnover stopped-flow and quench-flow assays. Cognate and noncognate reactions were measured using the enzymes and minimal biotin acceptor substrates from Escherichia coli, Pyrococcus horikoshii, and Homo sapiens. The kinetic analysis demonstrates that for all enzyme-substrate pairs the bimolecular rate of association of enzyme with substrate limits post-translational biotinylation. In addition, in noncognate reactions the three enzymes displayed a range of selectivities. These results highlight the importance of protein-protein binding kinetics for specific biotin addition to carboxylases and provide one mechanism for determining biotin distribution in metabolism.
Highlights
Grants R01-GM46511 and S10-RR15899. 1 To whom correspondence should be addressed
Oligomeric States of the Reacting Species in Biotin Transfer— Interpretation of kinetic and binding data requires knowledge of the assembly states of all species participating in a reaction
Biotin Transfer Rates to Cognate Substrates Are Similar for the Three Biotin Protein Ligases—In these experiments, BPL is preincubated with biotin at half of the enzyme concentration and excess ATP to allow for bio-5Ј-AMP synthesis
Summary
Chemicals and Buffers—All chemicals used in buffer preparation were at least reagent grade. To eliminate residual nucleic acid contamination PEI was added to the supernatant at a final concentration of 0.2% (w/v), the sample was stirred at 4 °C for 15 min, and the precipitate was removed by centrifugation at 12,000 rpm for 20 min. The pooled fractions (2 ml each) containing HsBCCP were concentrated and loaded at 0.1 ml/min onto a 150-ml S-100 Sephacryl resin (GE Healthcare) packed in a 1.5 ϫ 100-cm Econo-Column and equilibrated with 10 mM Tris, pH 7.5, at 4 °C, 0.8 M KCl, 5 mM 2-mercaptoethanol, 5% (v/v) glycerol. Reactions catalyzed by the human and E. coli enzyme were carried out in standard reaction buffer, which contains 10 mM Tris, pH 7.5, at 20 °C, 200 mM KCl, 2.5 mM MgCl2 Those reactions catalyzed by PhBPL were performed at pH 7.5, 40 °C, and a KCl concentration of 500 mM.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
