1ML3

Evidences for a flip-flop catalytic mechanism of Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase, from its crystal structure in complex with reacted irreversible inhibitor 2-(2-phosphono-ethyl)-acrylic acid 4-nitro-phenyl ester

Summary for 1ML3

• Entry DOI: 10.2210/pdb1ml3/pdb
• Related: 1A7K 1GYP
• Descriptor: Glyceraldehyde 3-phosphate dehydrogenase, glycosomal, NICOTINAMIDE-ADENINE-DINUCLEOTIDE, (3-FORMYL-BUT-3-ENYL)-PHOSPHONIC ACID, ... (4 entities in total)
• Functional Keywords: protein covalent-inhibitor complex, oxidoreductase
• Biological source: Trypanosoma cruzi
• Cellular location: Glycosome: P22513
• Total number of polymer chains: 4
• Total formula weight: 158932.72

Authors

Castilho, M.S.,Pavao, F.,Oliva, G. (deposition date: 2002-08-29, release date: 2003-07-08, Last modification date: 2024-10-16)

Primary citation

Castilho, M.S.,Pavao, F.,Oliva, G.,Ladame, S.,Willson, M.,Perie, J.
Evidence for the Two Phosphate Binding Sites of an Analogue of the Thioacyl Intermediate for the Trypanosoma cruzi Glyceraldehyde-3-phosphate Dehydrogenase-Catalyzed Reaction, from Its Crystal Structure.
Biochemistry, 42:7143-7151, 2003

PubMed Abstract: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the reversible oxidative phosphorylation of d-glyceraldehyde 3-phosphate (GAP) into d-glycerate 1,3-bisphosphate (1,3-diPG) in the presence of NAD(+) and inorganic phosphate (P(i)). Within the active site, two anion-binding sites were ascribed to the binding of the C3 phosphate of GAP (P(s)) and to the binding of the attacking phosphate ion (P(i)). The role played by these two sites in the catalytic mechanism in connection with the functional role of coenzyme exchange (NADH-NAD(+) shuttle) has been investigated by several studies leading to the C3 phosphate flipping model proposed by Skarzynski et al. [Skarzynski, T., Moody, P. C., and Wonacott, A. J. (1987) J. Mol. Biol. 193, 171-187]. This model has not yet received direct confirmation. To gain further insight into the role of both sites, we synthesized irreversible inhibitors which form with the essential cysteine residue a thioacyl enzyme analogue of the catalytic intermediate. Here we report the refined glycosomal Trypanosoma cruzi GAPDH in complex with a covalently bound GAP analogue at an improved resolution of 2.0-2.5 A. For this holo-thioacyl enzyme complex, a flip-flop movement is clearly characterized, the change from the P(i) to the P(s) binding site being correlated with the coenzyme exchange step: the weaker interaction of the intermediate when bound at the P(s) site with the cofactor allows its release and also the binding of the inorganic phosphate for the next catalytic step. This result gives strong experimental support for the generally accepted flip-flop model of the catalytic mechanism in GAPDH.

PubMed: 12795610
DOI: 10.1021/bi0206107

Experimental method

X-RAY DIFFRACTION (2.5 Å)