1JDE

K22A mutant of pyruvate, phosphate dikinase

Summary for 1JDE

• Entry DOI: 10.2210/pdb1jde/pdb
• Related: 1dik 1ggo 2dik
• Descriptor: PYRUVATE, PHOSPHATE DIKINASE, SULFATE ION (3 entities in total)
• Functional Keywords: transferase, phosphotransferase, kinase, nucleotide binding site mutant
• Biological source: Clostridium symbiosum
• Total number of polymer chains: 1
• Total formula weight: 96774.17

Authors

Ye, D.,Wei, M.,McGuire, M.,Huang, K.,Kapadia, G.,Herzberg, O.,Martin, B.M.,Dunaway-Mariano, D. (deposition date: 2001-06-13, release date: 2001-11-28, Last modification date: 2024-02-07)

Primary citation

Ye, D.,Wei, M.,McGuire, M.,Huang, K.,Kapadia, G.,Herzberg, O.,Martin, B.M.,Dunaway-Mariano, D.
Investigation of the catalytic site within the ATP-grasp domain of Clostridium symbiosum pyruvate phosphate dikinase.
J.Biol.Chem., 276:37630-37639, 2001

PubMed Abstract: 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.

PubMed: 11468288
DOI: 10.1074/jbc.M105631200

Experimental method

X-RAY DIFFRACTION (2.8 Å)