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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 esterEvidences 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
Structural highlights
FunctionEvolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedGlyceraldehyde-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. 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.,Castilho MS, Pavao F, Oliva G, Ladame S, Willson M, Perie J Biochemistry. 2003 Jun 17;42(23):7143-51. PMID:12795610[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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