Abstract
Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate. The reaction occurs in two separate catalytic domains, coupled by the long-range translocation of a biotinylated carrier domain (BCCP). Here, we use a series of hybrid PC enzymes to examine multiple BCCP translocation pathways in PC. These studies reveal that the BCCP domain of PC adopts a wide range of translocation pathways during catalysis. Furthermore, the allosteric activator, acetyl CoA, promotes one specific intermolecular carrier domain translocation pathway. These results provide a basis for the ordered thermodynamic state and the enhanced carboxyl group transfer efficiency in the presence of acetyl CoA, and reveal that the allosteric effector regulates enzyme activity by altering carrier domain movement. Given the similarities with enzymes involved in the modular synthesis of natural products, the allosteric regulation of carrier domain movements in PC is likely to be broadly applicable to multiple important enzyme systems.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Acetyl Coenzyme A / chemistry*
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Acetyl Coenzyme A / metabolism
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Allosteric Regulation
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Allosteric Site
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Amino Acid Sequence
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Aspartic Acid / chemistry*
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Aspartic Acid / metabolism
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Aspergillus nidulans / chemistry*
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Aspergillus nidulans / enzymology
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Biocatalysis
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Catalytic Domain
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Crystallography, X-Ray
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Gene Expression
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Kinetics
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Models, Molecular
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Protein Binding
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Protein Interaction Domains and Motifs
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Protein Multimerization
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Protein Structure, Quaternary
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Protein Structure, Secondary
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Protein Subunits / chemistry*
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Protein Subunits / genetics
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Protein Subunits / metabolism
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Pyruvate Carboxylase / chemistry*
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Pyruvate Carboxylase / genetics
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Pyruvate Carboxylase / metabolism
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Rhizobium etli / chemistry*
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Rhizobium etli / enzymology
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Substrate Specificity
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Thermodynamics
Substances
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Protein Subunits
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Recombinant Proteins
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Aspartic Acid
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Acetyl Coenzyme A
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Pyruvate Carboxylase