ABSTRACT
Pyruvate phosphate dikinase (PPDK) reversibly catalyzes the conversion of ATP, phosphate, and pyruvate into AMP, pyrophosphate, and phosphoenolpyruvate (PEP), respectively. Since the nucleotide binding site (in the N-terminal domain) and the pyruvate/PEP binding site (in the C-terminal domain) are separated by approximately 45 A, it has been proposed that an intermediary domain, called the central domain, swivels between these remote domains to transfer the phosphate. However, no direct structural evidence for the swiveling central domain has been found. In this study, the crystal structures of maize PPDK with and without PEP have been determined at 2.3 A resolution. These structures revealed that the central domain is located near the pyruvate/PEP binding C-terminal domain, in contrast to the PPDK from Clostridium symbiosum, wherein the central domain is located near the nucleotide-binding N-terminal domain. Structural comparisons between the maize and C. symbiosum PPDKs demonstrated that the swiveling motion of the central domain consists of a rotation of at least 92 degrees and a translation of 0.5 A. By comparing the maize PPDK structures with and without PEP, we have elucidated the mode of binding of PEP to the C-terminal domain and the induced conformational changes in the central domain.
Subject(s)
Plant Proteins/chemistry , Pyruvate, Orthophosphate Dikinase/chemistry , Thermodynamics , Zea mays/enzymology , Amino Acid Sequence , Binding Sites , Catalysis , Computer Simulation , Crystallization , Crystallography, X-Ray , Dimerization , Ligands , Models, Molecular , Molecular Sequence Data , Phosphoenolpyruvate/chemistry , Phosphoenolpyruvate/metabolism , Plant Proteins/metabolism , Protein Conformation , Protein Structure, Tertiary , Pyruvate, Orthophosphate Dikinase/metabolism , Substrate SpecificityABSTRACT
Pyruvate phosphate dikinase (PPDK) from maize catalyzes the reversible conversion of ATP, orthophosphate and pyruvate to AMP, pyrophosphate and PEP. In higher plants, this enzyme is believed to be involved in the C(4) dicarboxylic acid pathway. PPDK was crystallized by the vapour-diffusion method using polyethylene glycol as a precipitant. The crystals belong to the orthorhombic space group C2, with unit-cell parameters a = 108.2, b = 100.2, c = 108.4 A, beta = 96.5 degrees, and diffract to 2.3 A using SPring-8 synchrotron radiation.
