Abstract
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.
Highlights
The human lysosomal -hexosaminidases (EC 3.2.1.52) are composed of two structurally related subunits, ␣ and 
Much progress has been made in understanding the genetics and biochemistry of the hexosaminidases, details concerning the structures of the active sites and mechanisms of catalysis have only recently emerged
Using [3H]ATB-GalNAc as a photoaffinity label for the substrate binding site of -hexosaminidase B, Liessem et al [11] showed a specific incorporation into Glu-355 of the -subunit. This result, the strongest biochemical evidence to date in the identification of an active site, focused our attention on this evolutionarily conserved amino acid. We have extended these studies by analyzing mutant -subunits with Glu-355 converted into four other amino acids, Ala, Asp, Trp, and Gln
Summary
Vol 272, No 12, Issue of March 21, pp. 8002–8006, 1997 Printed in U.S.A. Evidence for the Involvement of Glu-355 in the Catalytic Action of Human -Hexosaminidase B*. The mutant -subunits lost catalytic activity but retained their ability to bind a substrate analogue, form homo- and heterodimers, function in the context of -hexosaminidase A in activator-dependent GM2 degradation, and undergo intracellular transport to lysosomes. These results, together with affinity labeling of Glu-355 and the recently described structure of an evolutionarily related chitobiase [12], provide compelling evidence that Glu-355 has an essential role in the catalytic mechanism of human -hexosaminidase B
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