A Substrate-induced Biotin Binding Pocket in the Carboxyltransferase Domain of Pyruvate Carboxylase

Abstract

Background: Biotin-dependent enzymes efficiently coordinate multiple reactions in physically separate active sites. Results: Substrate binding remodels the carboxyltransferase active site to form a biotin binding pocket. Conclusion: Pyruvate must bind before carboxybiotin can access the carboxyltransferase active site. Significance: The active sites of biotin-dependent enzymes control biotin access, providing a mechanism to coordinate the overall reaction between multiple active sites.

The role of biotin and oxamate in the carboxyltransferase reaction of pyruvate carboxylase

“…PC from Rhizobium etli ( Re PC) was previously subcloned into a modified pET-17b vector [20]. ΔBC Re PC [23] and ΔBCΔBCCP Re PC [11] were previously subcloned into a modified pET-28a vector.…”

“…Preparation and purification of the wild-type and mutant forms of Re PC, ΔBC Re PC and ΔBCΔBCCP Re PC were performed as previously described [11, 23]. All forms of Re PC and R eBCCP that do not contain the K1119Q mutation were > 95% biotinylated, as shown by SDS-PAGE analysis of an avidin-gel shift assay (Figure S1) [12].…”

“…Carboxybiotin, tethered to BCCP, enters the active site after pyruvate binds [11] and is decarboxylated to generate a biotin enolate intermediate [12]. A proton is subsequently shuttled from pyruvate to the biotin enolate via a conserved threonine residue, forming an enolpyruvate intermediate.…”

“…The description of oxaloacetate decarboxylation in PC is also plagued by contradictory literature reports concerning whether the biotin cofactor is essential for the decarboxylation reaction [11, 14, 16, 18, 19]. This is a crucial mechanistic question as it has significant implications on the stringency of the active site geometry and on the mode of carboxyl transfer.…”

“…Recent X-ray crystal structures of full-length PC [20, 21] have enabled new studies on the individual catalytic domains of the enzyme [11, 22]. Such studies serve to complement those of the full-length enzyme by simplifying the overall system, contributing to a more complete description of the PC reaction mechanism.…”