Identification of the active site nucleophile in the thermostable beta-glycosidase from the archaeon Sulfolobus solfataricus expressed in Escherichia coli.
Sulfolobus solfataricus beta-glycosidase expressed in Escherichia coli was fully inactivated at 65 degrees C, according to pseudo-first-order kinetics, by [3H]conduritol B epoxide (DL-1,2 anhydro-myo-inositol) synthesized as the active site directed inhibitor by a slight modification of Legler's procedure [Legler, G. (1977) Methods Enzymol. 46, 368-381]. The determination of kinetic constants for the inactivation showed that the process took place through the formation of a stabilized inhibitor-enzyme intermediate. Inactivation and reactivation studies suggested that the inhibitor-enzyme intermediate complex was formed more rapidly and hydrolyzed at a lower rate than it was for other glycosidases. Moreover, the stoichiometry of the binding, determined by electrospray mass spectrometric analysis, revealed that one molecule of the inhibitor was covalently bound to each enzyme subunit. The binding site for [3H]conduritol B epoxide was identified by the isolation and partial sequence analysis of the radioactive peptide obtained by cyanogen bromide and pepsin digests. Electrospray tandem mass analysis of the labeled peptide showed that the inhibitor was covalently bound to E387. This result, in agreement with data obtained from sequence alignments of S. solfataricus beta-glycosidase with other gluco- and galactosidases of the glycosyl hydrolase family 1 [Henrissat, B. (1991) Biochem. J. 280, 309-316], indicates that the conserved E387 is the nucleophilic amino acid residue in the active site of the enzyme.
Febbraio
F
F
Institute of Protein Biochemistry and Enzymology, CNR, Naples, Italy.
Barone
R
R
D'Auria
S
S
Rossi
M
M
Nucci
R
R
Piccialli
G
G
De Napoli
L
L
Orrù
S
S
Pucci
P
P
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D004926:Escherichia coli; D005959:Glucosidases; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007294:Inositol / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D016944:Sulfolobus / Q000201:enzymology*; D001617:beta-Glucosidase / Q000737:chemistry
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D007294:Inositol; D005959:Glucosidases; D006026:Glycoside Hydrolases; C104851:Sulfolobus solfataricus beta-glycosidase; D001617:beta-Glucosidase; C011423:conduritol epoxide