Characterization of the two anion-recognition sites of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus by site-directed mutagenesis and chemical modification

Abstract

The active site of the glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) contains two anion recognition sites which have been attributed to the phosphate binding of the substrates, namely, glyceraldehyde 3-phosphate (Ps site) and inorganic phosphate (Pi site) [Moras et al. (1975) J. Biol. Chem. 250, 9137-9162]. In order to probe the role of both sites during the catalytic event, Arg 195 from the Pi site and Arg 231 from the Ps site of the Bacillus stearothermophilus enzyme have been changed to Leu and Gly, respectively, by site-directed mutagenesis. A comparative study of the chemical reactivity of the mutants and wild type toward 2,3-butanedione revealed a similarly high reactivity only for the R195L mutant and wild type, suggesting that only Arg 231 is chemically reactive toward 2,3-butanedione and that its reactivity is not influenced by the presence of the residue Arg 195, which is only 4 A distant. The kinetic consequences of the mutations were also analyzed for the consecutive steps in the forward catalytic reaction. The replacement of Arg 195 by Leu leads to a marked decrease of the rate of the first steps of the reaction which lead to the acylenzyme formation, in particular, the rate of enzyme-substrate association, while these steps occur at a similar or higher rate when Arg 231 is replaced by Gly. Furthermore, the mutations R195L and R231G also result in a 550-fold and 16,400-fold decrease in the second-order rate constant of phosphorolysis. This step becomes rate-determining for the R195L mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Association of the Cytoplasmic Domain of Intercellular‐Adhesion Molecule‐1 with Glyceraldehyde‐3‐Phosphate Dehydrogenase and β‐Tubulin

“…3). The fact that this fragment also includes the NAD+-binding residue Asn313 [22] strenghtens the hypothesis of overlapping ICAM-1-binding and NAD+-binding sites.…”

“…The simplest interpretation of this result is that the NAD+-binding and ICAM-1-binding sites overlap. It is possible, however, that the conformational changes in GraP-DH triggered by NAD' [22] might destabilize the interaction between GraP-DH and the ICAM-1 C-terminal sequence.…”

“…The three-dimensional structure of the active site of the GraP-DH monomer has recently been described [22]. It is composed of catalytic residues (Cys149 and His176), two anion-binding sites for the inorganic phosphate ion (Arg195) and the substrate D-glyCeraldehyde 3-phosphate (Arg231) and the NAD+-binding domain (Asn313).…”

“…It is composed of catalytic residues (Cys149 and His176), two anion-binding sites for the inorganic phosphate ion (Arg195) and the substrate D-glyCeraldehyde 3-phosphate (Arg231) and the NAD+-binding domain (Asn313). It has been demonstrated [22] that binding of the adenosine moiety of NAD' to GraP-DH triggers conformational changes in GraP-DH which optimise the positioning of the catalytic residues and the anion-binding sites. Our observations that NAD' is able to elute GraP-DH (and its Ala229-Met325 fragment) from immobilized C27 ICAM-1, and that only the truncated peptide C15 ICAM-1 efficiently inhibits the NAD'-dependent glycolytic activity of GraP-DH, suggest that the ICAM-1-binding site may be constituted by at least two subsites.…”

“…Binding of the N-terminal residues of C27 and C23 ICAM-1 to subsite 1 might be inhibited by NAD', either by direct competition or due to conformational changes. Subsite 2, interacting with the IS C-terminal residues of ICAM-1 independently of NAD', might overlap with the catalytic site of the enzyme or be involved in the conformational changes in the active site which accompany NAD' stimulation of enzymic activity [22].…”