CHARACTERIZATION OF THE 2 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 dehydroge nase (GAPDH) contains two anion recognition sites which have been attr ibuted to the phosphate binding of the substrates, namely, glyceraldeh yde 3-phosphate (P-s site) and inorganic phosphate (P-i site) [Moras e t al. (1975) J. Biol. Chem. 250, 9137-9162]. In order to probe the rol e of both sites during the catalytic event, Arg 195 from the P-i site and Arg 231 from the P-s site of the Bacillus stearothermophilus enzym e have been changed to Leu and Gly, respectively, by site-directed mut agenesis. A comparative study of the chemical reactivity of the mutant s and wild type toward 2,3-butanedione revealed a similarly high react ivity only for the R195L mutant and wild type, suggesting that only Ar g 231 is chemically reactive toward 2,3-butanedione and that its react ivity is not influenced by the presence of the residue Arg 195, which is only 4 Angstrom 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 acyl enzyme formation, in particular, the rate of enzyme-substrate associat ion, while these steps occur at a similar or higher rate when Arg 231 is replaced by Gly. Furthermore, the mutations R195L and R231G also re sult in a 550-fold and 16 400-fold decrease in the second-order rate c onstant of phosphorolysis. This step becomes rate-determining for the R195L mutant. These results taken all together favor a reinterpretatio n of the individual contribution of the P-i and P-s sites [Moras et al . (1975) J. Biol. Chem. 250, 9137-9162] during the catalytic event as proposed by Wonacott and co-workers on the basis of B. stearothermophi lus GAPDH structure [Skarzynski et al. (1987) J. Mel. Biol. 193, 171-1 83]. During the steps preceding the formation of the acylenzyme interm ediate, the C-3 phosphate would first interact with the P-i site. The coenzyme exchange step could lead to a conformational isomerization of the acyl group, with the C-3 phosphate flipping from the P-i site to the P-s site. Phosphorolysis would then occur by attack of inorganic p hosphate with appropriate geometry from the Pi site. The proposed sche me would give a definite functional role to coenzyme exchange.