Evidence for the chemical activation of essential Cys-302 upon cofactor binding to nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans

Nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPN) from Str eptococcus mutans which catalyzes the irreversible oxidation of D-glycerald ehyde-3 phosphate (D-G3P) into 3-phosphoglycerate (3-PGA) in the presence o f NADP belongs to the aldehyde dehydrogenase (ALDK) superfamily. Oxidation of D-G3P into 3-PGA by GAPN involves the formation of a covalent enzyme int ermediate via the nucleophilic attack of the invariant Cys-302, Titration o f Cys-302 in the ape-enzyme by two different kinetic probes, iodoacetamide and 2,2'-dipyridyl disulfide, shows a pK(app) of 8.5 and a chemical reactiv ity surprisingly low compared to a reactive and accessible thiolate, Bindin g of NADP causes a strong increase of the reactivity of Cys-302-which is ti me dependent-with a pK(app) shift from 8.5 to 6.1, Concomitant with the inc rease in the Cys-302 reactivity, an additional protein fluorescence quenchi ng is observed. These data suggest that cofactor binding induces at least a local conformational rearrangement within the active site. The efficiency of the rearrangement depends on the structure of the cofactors and on the p rotonation of an amino acid with a pK(app) of 5.7. The rate of the rearrang ement also strongly increases when temperature decreases. The data on the c onformational rearrangement also reveal an amino acid with a pK(app) of 7.6 whose deprotonation increases the reactivity of the thiolate of Cys-302 by a 3-fold factor. The nature of the amino acid involved-which should be loc ated close to Cys-302 in the hole-active form-is likely the invariant Glu-2 68. Changing Glu-268 into Ala or Cys-302 into Ala leads to mutants in which the rearrangement is only efficient in the presence of saturating concentr ations of both NADP and G3P. The structural aspects of the conformational r earrangement occurring during the catalytic process in the wild-type GAPN s hould include at least reorientation of both Cys-302 and Glu-268 side chain s and repositioning of the nicotinamide ring of the cofactor to permit the chemical activation of Cys-302 and the formation of an efficient ternary co mplex. Thus, it is likely that the conformation of the active site in the r eported X-ray structures of ALDHs determined so far in the presence of cofa ctor, in which the side chains of Cys-302 and Glu-268 are 6.7 Angstrom apar t from each other, does not represent the biological active form.