EVIDENCE FOR THE INVOLVEMENT OF GLU-355 IN THE CATALYTIC ACTION OF HUMAN BETA-HEXOSAMINIDASE-B

In a previous study the photoactivable affinity probe, 1-[([6-H-3]2-ac etamido-2-deoxy-1-beta-D-galactopyr anosyl)thio]-butane, was used to i dentify the active site of beta-hexosaminidase B, a beta-subunit dimer (Liessem, B., Glombitza, G. J., Knell, F., Lehmann, J., Kellermann, J ., Lottspeich, F., and Sandhoff, K. (1995) J. Biol. Chem. 270, 23693-2 3699). The probe predominately labeled Glu-355, a highly conserved res idue among hexosaminidases. To determine if Glu-355 has a role in cata lysis, beta-subunit mutants were prepared with the Glu-355 codon alter ed to either Ala, Gln, Asp, or Trp. After expression of mutant protein s using recombinant baculovirus, the enzyme activity associated with t he beta-subunits was found to be reduced to background levels. Althoug h catalytic activity was lost, the mutations did not otherwise affect the folding or assembly of the subunits. The mutant beta-subunits coul d be isolated using substrate affinity chromatography, indicating they contained intact substrate binding sites. As shown by cross-linking w ith di-succinimidyl suberate, the mutant beta-subunits were properly a ssembled. They could also participate in the formation of functional b eta-hexosaminidase A activity as indicated by activator-dependent G(M2 ) ganglioside degradation activity produced by co-expression of the mu tant beta-subunits with the cu-subunit. Finally, the mutant subunits s howed normal lysosomal processing in COS-1 cells, demonstrating that a transport-competent protein conformation had been attained. Collectiv ely the results provide strong support for the intimate involvement of Glu-355 in beta-hexosaminidase B-mediated catalysis.