Acid-base catalysis in Leuconostoc mesenteroides sucrose phosphorylase probed by site-directed mutagenesis and detailed kinetic comparison of wild-type and Glu-237->Gln mutant enzymes

Alexandra Schwarz , Lothar Brecker , Bernd Nidetzky (1)

1 TU Graz - Graz University of Technology [Graz]

The role of acid-base catalysis in the two-step enzymatic mechanism of {alpha}-retaining glucosyl transfer by Leuconostoc mesenteroides sucrose phosphorylase has been examined through site-directed replacement of the putative catalytic Glu-237 and detailed comparison of purified wild-type and Glu-237->Gln mutant enzymes using steady-state kinetics. Reactions with substrates requiring Brønsted catalytic assistance for glucosylation or deglucosylation were selectively slowed at the respective step, about 10 5}-fold, in E237Q. Azide, acetate and formate but not halides restored catalytic activity, up to 300-fold, in E237Q under conditions in which the deglycosylation step was rate-determining, and promoted production of the corresponding {alpha}-glucosides. In situ proton NMR studies of the chemical rescue of E237Q by acetate and formate revealed that enzymatically formed {alpha}-glucose 1-esters decomposed spontaneously via acyl group migration and hydrolysis. Using pH profiles of k cat}/K m}, the pH dependences of kinetically isolated glucosylation and deglucosylation steps were analyzed for wild-type and E237Q. Glucosylation of the wild-type proceeded optimally above and below apparent pK a} values of about 5.6 and 7.2 respectively whereas deglucosylation was dependent on the apparent single ionization of a group of pK a} ~ 5.8 that must be deprotonated for reaction. Glucosylation of E237Q was slowed below apparent pK a} ~ 6.0 but had lost the high pH dependence of the wild-type. Deglucosylation of E237Q was pH-independent. The results allow unequivocal assignment of Glu-237 as the catalytic acid/base of sucrose phosphorylase. They support a mechanism in which the pK a} of Glu-237 cycles between ~ 7.2 in free enzyme to ~ 5.8 in glucosyl enzyme intermediate, ensuring optimal participation of the Glu side chain at each step in catalysis. Enzyme deglucosylation to an anionic nucleophile took place with Glu-237 protonated or unprotonated. The results delineate how conserved active-site groups of retaining glycoside hydrolases can accommodate enzymatic function of a phosphorylase.

Mots clés: Life Sciences

Alexandra Schwarz, Lothar Brecker, Bernd Nidetzky. Acid-base catalysis in Leuconostoc mesenteroides sucrose phosphorylase probed by site-directed mutagenesis and detailed kinetic comparison of wild-type and Glu-237->Gln mutant enzymes. Biochemical Journal, 2007, 403 (3), pp.441-449. ⟨10.1042/BJ20070042⟩. ⟨hal-00478733⟩