A new chemical mechanism catalyzed by a mutated aldehyde dehydrogenase.
NAD(P) aldehyde dehydrogenases (EC 1.2.1.3) are a family of enzymes that oxidize a wide variety of aldehydes into acid or activated acid compounds. Using site-directed mutagenesis, the essential nucleophilic Cys 149 in the NAD-dependent phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Escherichia coli has been replaced by alanine. Not unexpectedly, the resulting mutant no longer shows any oxidoreduction phosphorylating activity. The same mutation, however, endows the enzyme with a novel oxidoreduction nonphosphorylating activity, converting glyceraldehyde 3-phosphate into 3-phosphoglycerate. Our study further provides evidence for an alternative mechanism in which the true substrate is the gem-diol entity instead of the aldehyde form. This implies that no acylenzyme intermediate is formed during the catalytic event. Therefore, the mutant C149A is a new enzyme which catalyzes a distinct reaction with a chemical mechanism different from that of its parent phosphorylating glyceraldehyde-3-phosphate dehydrogenase. This finding demonstrates the possibility of an alternative route for the chemical reaction catalyzed by classical nonphosphorylating aldehyde dehydrogenases.
Corbier
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C
Laboratoire d'Enzymologie et de Génie Génétique, Université de Nancy I, URA CNRS 457, Vandoeuvre-lès-Nancy. France.
Della Seta
F
F
Branlant
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G
D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed
D005987:Glyceraldehyde-3-Phosphate Dehydrogenases