Corbier C, Della Seta F, Branlant G
Laboratoire d'Enzymologie et de Génie Génétique, Université de Nancy I, URA CNRS 457, Vandoeuvre-lès-Nancy. France.
Biochemistry. 1992 Dec 15;31(49):12532-5. doi: 10.1021/bi00164a033.
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Cys 149 in the NAD-dependent phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Escherichia coli has been replaced by alanine.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.
NAD(P)醛脱氢酶（EC 1.2.1.3）是一类将多种醛氧化为酸或活化酸化合物的酶。通过定点诱变，来自大肠杆菌的依赖NAD的磷酸化甘油醛-3-磷酸脱氢酶中必需的亲核半胱氨酸149已被丙氨酸取代。不出所料，所得突变体不再表现出任何氧化还原磷酸化活性。然而，相同的突变赋予该酶一种新的氧化还原非磷酸化活性，即将甘油醛-3-磷酸转化为3-磷酸甘油酸。我们的研究进一步为一种替代机制提供了证据，其中真正的底物是偕二醇实体而非醛形式。这意味着在催化过程中不会形成酰基酶中间体。因此，突变体C149A是一种新酶，它催化一种与亲本磷酸化甘油醛-3-磷酸脱氢酶不同的化学反应机制的独特反应。这一发现证明了经典非磷酸化醛脱氢酶催化的化学反应存在替代途径的可能性。
