1jde

From Proteopedia

---

Revision as of 16:00, 20 November 2007

proteopedia linkproteopedia link

spinqualitylabelsall models 1jde, resolution 2.8Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

K22A mutant of pyruvate, phosphate dikinase

Overview

Pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of ATP, P(i), and pyruvate with AMP, PP(i), and phosphoenolpyruvate (PEP) in three, partial reactions as follows: 1) E-His + ATP --> E-His-PP.AMP; 2), E-His-PP.AMP + P(i) --> E-His-P.AMP.PP(i); and 3) E-His-P + pyruvate -->, E.PEP using His-455 as the carrier of the transferred phosphoryl groups., The crystal structure of the Clostridium symbiosum PPDK (in the unbound, state) reveals a three-domain structure consisting of consecutive, N-terminal, central His-455, and C-terminal domains. The N-terminal and, central His-455 domains catalyze partial reactions 1 and 2, whereas the, C-terminal and central His-455 domains catalyze partial reaction 3., Attempts to obtain a crystal structure of the enzyme with substrate, ligands bound at the nucleotide binding domain have been unsuccessful. The, object of the present study is to demonstrate Mg(II) activation of, catalysis at the ATP/P(i) active site, to identify the residues at the, ATP/P(i) active site that contribute to catalysis, and to identify roles, for these residues based on their positions within the active site, scaffold. First, Mg(II) activation studies of catalysis of E + ATP + P(i), --> E-P + AMP + PP(i) partial reaction were carried out using a truncation, mutant (Tem533) in which the C-terminal domain is absent. The kinetics, show that a minimum of 2 Mg(II) per active site is required for the, reaction. The active site residues used for substrate/cofactor, binding/activation were identified by site-directed mutagenesis. Lys-22, Arg-92, Asp-321, Glu-323, and Gln-335 mutants were found to be inactive;, Arg-337, Glu-279, Asp-280, and Arg-135 mutants were partially active; and, Thr-253 and Gln-240 mutants were almost fully active. The participation of, the nucleotide ribose 2'-OH and alpha-P in enzyme binding is indicated by, the loss of productive binding seen with substrate analogs modified at, these positions. The ATP, P(i), and Mg(II) ions were docked into the PPDK, N-terminal domain crevice, in an orientation consistent with, substrate/cofactor binding modes observed for other members of the, ATP-Grasp fold enzyme superfamily and consistent with the, structure-function data. On the basis of this docking model, the ATP, polyphosphate moiety is oriented/activated for pyrophosphoryl transfer, through interaction with Lys-22 (gamma-P), Arg-92 (alpha-P), and the, Gly-101 to Met-103 loop (gamma-P) as well as with the Mg(II) cofactors., The P(i) is oriented/activated for partial reaction 2 through interaction, with Arg-337 and a Mg(II) cofactor. The Mg(II) ions are bound through, interaction with Asp-321, Glu-323, and Gln-335 and substrate. Residues, Glu-279, Asp-280, and Arg-135 are suggested to function in the closure of, an active site loop, over the nucleotide ribose-binding site.

About this Structure

1JDE is a Single protein structure of sequence from Clostridium symbiosum with SO4 as ligand. Active as Pyruvate, phosphate dikinase, with EC number 2.7.9.1 Full crystallographic information is available from OCA.

Reference

Investigation of the catalytic site within the ATP-grasp domain of Clostridium symbiosum pyruvate phosphate dikinase., Ye D, Wei M, McGuire M, Huang K, Kapadia G, Herzberg O, Martin BM, Dunaway-Mariano D, J Biol Chem. 2001 Oct 5;276(40):37630-9. Epub 2001 Jul 23. PMID:11468288

Page seeded by OCA on Tue Nov 20 18:08:07 2007