Characterization of the two anion-recognition sites of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus by site-directed mutagenesis and chemical modification.
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)
Corbier
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Laboratoire d'Enzymologie et de Génie Génétique, Université de Nancy I, URA CNRS 457, Vandoeuvre-les-Nancy, France.
Michels
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Wonacott
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Branlant
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D000838:Anions; D001665:Binding Sites; D003460:Crystallization; D003931:Diacetyl / Q000494:pharmacology; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism
D000838:Anions; D010710:Phosphates; D009243:NAD; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D003931:Diacetyl