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.

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.

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.

[Synthesis of 2-acetamido-1,4-imino-1,2,4-tridesoxy-D-galactitol and competitive inhibition of human lysosomal 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.