Swiveling domain mechanism in pyruvate phosphate dikinase.

Printer-friendly versionPrinter-friendly versionPDF versionPDF version
TitleSwiveling domain mechanism in pyruvate phosphate dikinase.
Publication TypeJournal Article
Year of Publication2007
AuthorsLim, K, Read, RJ, Chen, CCH, Tempczyk, A, Wei, M, Ye, D, Wu, C, Dunaway-Mariano, D, Herzberg, O
JournalBiochemistry
Volume46
Issue51
Pagination14845-53
Date Published2007 Dec 25
ISSN0006-2960
KeywordsBinding Sites, Clostridium symbiosum, Crystallography, X-Ray, Isoenzymes, Kinetics, Mutation, Nucleotides, Protein Structure, Quaternary, Protein Structure, Tertiary, Pyruvate, Orthophosphate Dikinase, Structural Homology, Protein
Abstract

Pyruvate phosphate dikinase (PPDK) catalyzes the reversible conversion of phosphoenolpyruvate (PEP), AMP, and Pi to pyruvate and ATP. The enzyme contains two remotely located reaction centers: the nucleotide partial reaction takes place at the N-terminal domain, and the PEP/pyruvate partial reaction takes place at the C-terminal domain. A central domain, tethered to the N- and C-terminal domains by two closely associated linkers, contains a phosphorylatable histidine residue (His455). The molecular architecture suggests a swiveling domain mechanism that shuttles a phosphoryl group between the two reaction centers. In an early structure of PPDK from Clostridium symbiosum, the His445-containing domain (His domain) was positioned close to the nucleotide binding domain and did not contact the PEP/pyruvate-binding domain. Here, we present the crystal structure of a second conformational state of C. symbiosum PPDK with the His domain adjacent to the PEP-binding domain. The structure was obtained by producing a three-residue mutant protein (R219E/E271R/S262D) that introduces repulsion between the His and nucleotide-binding domains but preserves viable interactions with the PEP/pyruvate-binding domain. Accordingly, the mutant enzyme is competent in catalyzing the PEP/pyruvate half-reaction but the overall activity is abolished. The new structure confirms the swivel motion of the His domain. In addition, upon detachment from the His domain, the two nucleotide-binding subdomains undergo a hinge motion that opens the active-site cleft. A similar hinge motion is expected to accompany nucleotide binding (cleft closure) and release (cleft opening). A model of the coupled swivel and cleft opening motions was generated by interpolation between two end conformations, each with His455 positioned for phosphoryl group transfer from/to one of the substrates. The trajectory of the His domain avoids major clashes with the partner domains while preserving the association of the two linker segments.

DOI10.1021/bi701848w
Alternate JournalBiochemistry
PubMed ID18052212
Grant ListR01GM36260 / GM / NIGMS NIH HHS / United States