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A novel role of the conserved glutamate in aldehyde dehydrogenase catalysis
Author(s) -
Tsybovsky Yaroslav,
Krupenko Sergey A.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1012.3
Subject(s) - chemistry , active site , aldehyde dehydrogenase , acylation , nad+ kinase , cofactor , stereochemistry , cysteine , alanine , glutamate dehydrogenase , enzyme , biochemistry , coenzyme a , residue (chemistry) , catalysis , amino acid , glutamate receptor , receptor , reductase
Aldehyde dehydrogenase (ALDH) catalysis requires the NAD(P) + coenzyme and involves two steps, acylation and deacylation. The active site of every ALDH has two strictly conserved residues, Cys302 and Glu268, which are essential for catalysis. While the role of the cysteine as a catalytic nucleophile is well established, the function of the glutamate is ambiguous. It has been proposed that the glutamate either deprotonates Cys302 during the acylation step or activates a water molecule in the deacylation step. Our crystallographic studies, aimed at clarifying the role of the glutamate, revealed that the substitution of an alanine for this residue in the ALDH domain of rat 10‐formyltetrahydrofolate dehydrogenase traps NADP + in the active site, rendering the enzyme inactive. In this mutant, the coenzyme is bound so tightly that it cannot be removed by conventional techniques such as dialysis or chromatography, as well as by back‐soaking of protein crystals. At the same time, the conformation of the coenzyme remains the same as in the wild‐type enzyme. Thus, the glutamate is apparently required to prevent irreversible binding of NADP + and to provide coenzyme mobility that is necessary for ALDH catalysis.