Evidence for the involvement of Glu-355 in the catalytic action of human beta-hexosaminidase B.

In a previous study the photoactivable affinity probe, 3-azi-1-[([6-3H]2-acetamido-2-deoxy-1-beta-D-galactopyranosyl)thio ]-b utane, was used to identify the active site of beta-hexosaminidase B, a beta-subunit dimer (Liessem, B., Glombitza, G. J., Knoll, F., Lehmann, J., Kellermann, J., Lottspeich, F., and Sandhoff, K. (1995) J. Biol. Chem. 270, 23693-23699). The probe predominately labeled Glu-355, a highly conserved residue among hexosaminidases. To determine if Glu-355 has a role in catalysis, beta-subunit mutants were prepared with the Glu-355 codon altered to either Ala, Gln, Asp, or Trp. After expression of mutant proteins using recombinant baculovirus, the enzyme activity associated with the beta-subunits was found to be reduced to background levels. Although catalytic activity was lost, the mutations did not otherwise affect the folding or assembly of the subunits. The mutant beta-subunits could be isolated using substrate affinity chromatography, indicating they contained intact substrate binding sites. As shown by cross-linking with disuccinimidyl suberate, the mutant beta-subunits were properly assembled. They could also participate in the formation of functional beta-hexosaminidase A activity as indicated by activator-dependent GM2 ganglioside degradation activity produced by co-expression of the mutant beta-subunits with the alpha-subunit. Finally, the mutant subunits showed normal lysosomal processing in COS-1 cells, demonstrating that a transport-competent protein conformation had been attained. Collectively the results provide strong support for the intimate involvement of Glu-355 in beta-hexosaminidase B-mediated catalysis.

Identification of domains in human beta-hexosaminidase that determine substrate specificity.

The lysosomal beta-hexosaminidases are dimers composed of alpha and beta subunits. beta-Hexosaminidase A (alphabeta) is a heterodimer, whereas hexosaminidase B (betabeta) and S (alphaalpha) are homodimers. Although containing a high degree of amino acid identity, each subunit expresses a unique active site that can be distinguished by a differential ability to hydrolyze charged substrates. The site on the beta-subunit primarily degrades neutral substrates, whereas the alpha-subunit site is, in addition, active against sulfated substrates. Isozyme specificity is also exhibited with glycolipid substrates. Among human isozymes, only beta-hexosaminidase A together with the GM2 activator protein can degrade the natural substrate, GM2 ganglioside, at physiologically significant rates. To identify the domains of the human beta-hexosaminidase subunits that determine substrate specificity, we have generated chimeric subunits containing both alpha- and beta-subunit sequences. The chimeric constructs were expressed in HeLa cells to screen for activity and then selected constructs were produced in the baculovirus expression system to assess their ability to degrade GM2 ganglioside in the presence of GM2 activator protein. Generation of activity against the sulfated substrate required the substitution of two noncontinuous alpha-subunit sequences (amino acids 1-191 and 403-529) into analogous positions of the beta-subunit. Chimeric constructs containing only one of these regions linked to the beta-subunit sequence showed either neutral substrate activity only (amino acids 1-191) or lacked enzyme activity entirely (amino acids 403-529). Neither the chimeras nor the wild-type subunits displayed activator-dependent GM2-hydrolyzing activity when expressed alone. However, one chimeric subunit containing alpha amino acids 1-191 fused with beta amino acids 225 to 556, when co-expressed with the wild-type alpha-subunit, showed activity comparable with that of recombinant beta-hexosaminidase A formed by the co-expression of the alpha- and beta-subunits. This result indicates that the beta-subunit amino acids 225-556 contribute an essential function in the GM2-hydrolyzing activity of beta-hexosaminidase A.

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.

[Synthesis of 2-acetamido-1,4-imino-1,2,4-tridesoxy-D-galactitol and competitive inhibition of human lysosomal beta-hexosaminidase A]. / Synthese von 2-Acetamido-1,4-imino-1,2,4-tridesoxy-D-galaktitol und kompetitive Inhibition der humanen lysosomalen beta-Hexosaminidase A.

The synthesis of 2-acetamido-1,4-imino-1,2,4-trideoxy-D-galactitol (1; 2-acetamido-4-amino-1,4-anhydro-2,4-dideoxy-D-galactitol) by two different routes starting from 2-acetamido-2-deoxy-D-glucose is described. Compound 1 is a competitive inhibitor of human lysosomal beta-hexosaminidase A with K(i) values of 18 microM (beta-subunit) and 220 microM (alpha-subunit). Similar properties were found for the already known 2-acetamido-2-deoxy-D-gluco-hydroximo-1,4-lactone.