Center for Advanced Research In Biotechnology, University of Maryland Biotechnology Institute, Rockville, MD 20850, USA
Pyruvate phosphate dikinase (PPDK) catalyzes the reversible conversion of ATP, inorganic phosphate and pyruvate to AMP, pyrophosphate and phosphoenolpyruvate (PEP). The enzyme is a large dimeric molecule (monomer molecular mass of ~96,000 Dalton). Its mechanism involves three phosphoryl group transfer reactions that are mediated by a histidine residue. The crystal structure of PPDK consists of three-domains with the phospho-histidine domain flanked by the nucleotide and the PEP/pyruvate domains, with the two substrate binding sites about 45 degrees apart. A search through the structures available in the Protein Data Bank revealed that despite the lack of amino-acid sequence homology, the fold of the nucleotide binding domain resembles that of a number of ADP-utilizing proteins, and the alpha/beta barrel of the PEP/pyruvate domain resembles most closely that of pyruvate kinase. The modes of substrate binding have been deduced by analogy to D-Ala:D-Ala ligase and to pyruvate kinase, demonstrating another use for the data base. The mechanism of the enzyme may now be understood in light of these binding modes. Coupling between the two remote active sites is facilitated by two conformational states of the phospho-histidine domain. While the crystal structure represents the state of interaction with the nucleotide binding domain, the second state is achieved by swiveling around two flexible peptide linkers. This dramatic conformational transition brings the phospho-carrier residue in close proximity to PEP/pyruvate. The swiveling-domain paradigm provides an effective mechanism for communication in complex multi-domain/multi active site proteins.