Photoaffinity labeling of human lysosomal beta-hexosaminidase B. Identification of Glu-355 at the substrate binding site.
The carbene precursor 3-azi-1-[([6-3H]-2-acetamido-2-deoxy-1-beta-D-galactopyranosyl)thi o -butane (also designated [3H]-1-ATB-GalNAc) has been used as a photoaffinity label for human lysosomal beta-hexosaminidase B (Hex B, EC 3.2.1.52) purified to apparent homogeneity from postmortal liver. [3H]-1-ATB-GalNAc behaved as an active site-directed inhibitor, which bound covalently to Hex B upon photolysis at 350 nm and resulted in 15% inactivation of enzyme activity. Up to 75% of the inactivation of Hex B was prevented by including the competitive inhibitor 2-acetamido-2-deoxy-D-glucono-1,5-lactone in the photoaffinity experiment. Incubation of [3H]-1-ATB-GalNAc with the enzyme followed by irradiation and subsequent separation of the three polypeptides composing the beta-subunit led mainly to labeling of the beta a-polypeptide. Subsequent proteolysis of beta a with trypsin and separation of the resulting peptides by high pressure liquid chromatography yielded one prominently labeled peptide fraction. Edman degradation resulted in the sequence E339ISEVFPDQFIHLGGD-EVEFK359. However, no modified amino acid was detected, indicating that the photoaffinity label was presumably bound to the peptide by a labile ester linkage. This was proven when the radiolabel was almost completely released from the peptide by treatment with aqueous ammonium hydroxide. Simultaneously, Glu-355 was converted into Gln-355, which is located within a region of Hex B that shows considerable homology with the alpha-subunit of human hexosaminidase A and other hexosaminidases from various species.
Liessem
B
B
Institut für Organische Chemie und Biochemie, Universität Bonn, Federal Republic of Germany.
Glombitza
G J
GJ
Knoll
F
F
Lehmann
J
J
Kellermann
J
J
Lottspeich
F
F
Sandhoff
K
K
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001391:Azo Compounds; D001665:Binding Sites; D002240:Carbohydrate Sequence; D005678:G(M2) Ganglioside / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D006017:Glycolipids / Q000737:chemistry; D054818:Hexosaminidase A; D054819:Hexosaminidase B; D006801:Humans; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology; D008247:Lysosomes / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013865:Thioglycosides; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000345:Affinity Labels; D001391:Azo Compounds; D006017:Glycolipids; D010446:Peptide Fragments; D013865:Thioglycosides; C076591:3-azi-1-((2-acetamido-2-deoxy-1-galactopyranosyl)thio)butane; D005678:G(M2) Ganglioside; D018698:Glutamic Acid; D054818:Hexosaminidase A; D054819:Hexosaminidase B; D001619:beta-N-Acetylhexosaminidases