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.[1]References
- Photoaffinity labeling of human lysosomal beta-hexosaminidase B. Identification of Glu-355 at the substrate binding site. Liessem, B., Glombitza, G.J., Knoll, F., Lehmann, J., Kellermann, J., Lottspeich, F., Sandhoff, K. J. Biol. Chem. (1995) [Pubmed]
Annotations and hyperlinks in this abstract are from individual authors of WikiGenes or automatically generated by the WikiGenes Data Mining Engine. The abstract is from MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.About WikiGenesOpen Access LicencePrivacy PolicyTerms of Useapsburg