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)

The two anion-binding sites of the glycolytic glyceraldehydc-3-pl~osphalc dehydrogenase (Craf -nli), thc Ps and Pi sites, were originally proposed by Moras ct al. IMoras mnphilus Graf-DH were constructed by site-directed mutagenesis and their kinetic properties were detcrmined and compared with rhose of mutants R19SL and K23 1G, alreiidy described ICorbier, C., Michels, S., Wonacott, A. & Bmnlant, G. (1994) Biochetnistry 33, 3260-32651. Taking advantage of the opportunity to study both the oxidoreduction and the phosphorylation step independently and the fact that the phosphorylation becomes ratc detertilining for most of the muratits, the relative energetic contribution of each mutated amino acid to the phosphorylation step was evaluated. It was concluded that (a) Ps amino acids contribute more than the Pi amino acids to the stabilisation of the transition state relative to thu ground state and (b) the side chain of arginine contributes iiiore than that of the thrconiilc residue. It was also concluded that the differences observed in the efficiuncy of the phc-isphorylation step for Ps and Pi mutants is a consequence of the oricntation of the thioeskr bond of the thioacyl intermediatc relative to the attacking inorganic phosphate end not of a change in the intrinsic electrophilic property of the thioacyl intermediate. Furthermore, the kinetic results on thc overall steps leading to the acyl-enzyme t'ormation provided supplementary evidcnce that the C3 phosphate moiety of the glyceraldehyde 3-phosphate in-

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Non-phosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.9) from spinach leaves was purified to homogeneity using an improved purification procedure. Thus, a major contaminant with molecular mass and ion-exchange properties similar to non-phosphorylating GAPDH was eliminated. Using this pure non-phosphorylating GAPDH, cofactor stereospecificity was determined by 'H NMR. Analysis of the NADPH formed from the hydride transfer from glyceraldehyde-3-phosphate to [4-*H]NADP showed that the enzyme belongs to the A-stereospecific dehydrogenase family. This stereospecificity is the same as that described for the aldehyde dehydrogenase (ALDH) superfamily and opposite to that of the phosphorylating GAPDH. Moreover, results from peptide sequencing analysis suggest a similarity in sequence between the non-phosphorylating GAPDH and ALDHs. Thus, the results taken all together strongly suggest that non-phosphorylating GAPDH belongs to the ALDH family and has no close relationship to the phosphorylating GAPDH class.