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 cycloamylose synthesis reactions. Family 57 glycoside hydrolases have not been well investigated, and even the catalytic mechanism involving the active site residues has not been studied. Using 3-ketobutylidene-beta-2-chloro-4-nitrophenyl 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-enzyme intermediate, in which a donor substrate was covalently bound to the catalytic nucleophile, was trapped by treating the enzyme with 3KBG5CNP in the absence of an acceptor and was detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry after peptic digestion. Postsource decay analysis suggested that either Glu-123 or Glu-129 was the catalytic nucleophile of TLGT. Glu-123 was completely conserved between family 57 enzymes, and the catalytic activity of the E123Q mutant enzyme was greatly decreased. On the other hand, Glu-129 was a variable residue, and the catalytic activity of the E129Q mutant enzyme was not decreased. These results indicate that Glu-123 is the catalytic nucleophile of TLGT. Sequence alignment 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 glycoside hydrolases may have had a common ancestor.
Imamura
H
H
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Fushinobu
S
S
Jeon
B S
BS
Wakagi
T
T
Matsuzawa
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D002240:Carbohydrate Sequence; D020134:Catalytic Domain / Q000235:genetics; D005960:Glucosides / Q000737:chemistry; D006004:Glycogen Debranching Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics
D005960:Glucosides; D006004:Glycogen Debranching Enzyme System; C071715:3-ketobutylidene 2-chloro-4-nitrophenylmaltopentaoside; C020569:4 alpha-glucanotransferase