Identification of the catalytic residue of Thermococcus litoralis 4-alpha-glucanotransferase through mechanism-based labeling

Thermococcus litoralis 4-alpha -glucanotransferase (TLGT) belongs to family 57 of glycoside hydrolases and catalyzes the disproportionation and cycloa mylose synthesis reactions. Family 57 glycoside hydrolases have not been we ll investigated, and even the catalytic mechanism involving the active site residues has not been studied. Using 3-ketobutyhdene-beta -2-cliloro-4-nit rophenyl maltopentaoside (3KBG5CNP) as a donor and glucose as an acceptor, we showed that the disproportionation reaction of TLGT involves a ping-pong bi-bi mechanism. On the basis of this reaction mechanism, the glycosyl-enz yme intermediate, in which a donor substrate was covalently bound to the ca talytic nucleophile, was trapped by treating the enzyme with 3KBG5CNP in th e absence of an acceptor and was detected by matrix-assisted laser desorpti on ionization time-of-flight mass spectrometry after peptic digestion. Post source decay analysis suggested that either Glu-123 or Glu-129 was the cata lytic nucleophile of TLGT. Glu-123 was completely conserved between family 57 enzymes, and the catalytic activity of the E123Q mutant enzyme was great ly decreased. On the other hand, Glu-129 was a variable residue, and the ca talytic activity of the E129Q mutant enzyme was not decreased. These result s indicate that Glu-123 is the catalytic nucleophile of TLGT. Sequence alig nment of TLGT and family 38 enzymes (class II alpha -mannosidases) revealed that Glu-123 of TLGT corresponds to the nucleophilic aspartic acid residue of family 38 glycoside hydrolases, suggesting that family 57 and 38 glycos ide hydrolases may have had a common ancestor.