Faculty Collaboration Database

A substrate-induced biotin binding pocket in the carboxyltransferase domain of pyruvate carboxylase. J Biol Chem 2013 Jul 05;288(27):19915-25

Date

05/24/2013

Pubmed ID

23698000

Pubmed Central ID

PMC3707692

DOI

10.1074/jbc.M113.477828

Scopus ID

2-s2.0-84880051370 (requires institutional sign-in at Scopus site)   20 Citations

Abstract

Biotin-dependent enzymes catalyze carboxyl transfer reactions by efficiently coordinating multiple reactions between spatially distinct active sites. Pyruvate carboxylase (PC), a multifunctional biotin-dependent enzyme, catalyzes the bicarbonate- and MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To complete the overall reaction, the tethered biotin prosthetic group must first gain access to the biotin carboxylase domain and become carboxylated and then translocate to the carboxyltransferase domain, where the carboxyl group is transferred from biotin to pyruvate. Here, we report structural and kinetic evidence for the formation of a substrate-induced biotin binding pocket in the carboxyltransferase domain of PC from Rhizobium etli. Structures of the carboxyltransferase domain reveal that R. etli PC occupies a symmetrical conformation in the absence of the biotin carboxylase domain and that the carboxyltransferase domain active site is conformationally rearranged upon pyruvate binding. This conformational change is stabilized by the interaction of the conserved residues Asp(590) and Tyr(628) and results in the formation of the biotin binding pocket. Site-directed mutations at these residues reduce the rate of biotin-dependent reactions but have no effect on the rate of biotin-independent oxaloacetate decarboxylation. Given the conservation with carboxyltransferase domains in oxaloacetate decarboxylase and transcarboxylase, the structure-based mechanism described for PC may be applicable to the larger family of biotin-dependent enzymes.

Author List

Lietzan AD, St Maurice M

Author

Martin St. Maurice PhD Associate Professor in the Biology department at Marquette University

MESH terms used to index this publication - Major topics in bold

Bacterial Proteins
Binding Sites
Biotin
Carboxyl and Carbamoyl Transferases
Mutagenesis, Site-Directed
Protein Structure, Tertiary
Pyruvate Carboxylase
Pyruvic Acid
Rhizobium etli