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.
Pennybacker
M
M
Section on Biochemical Genetics, Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
Schuette
C G
CG
Liessem
B
B
Hepbildikler
S T
ST
Kopetka
J A
JA
Ellis
M R
MR
Myerowitz
R
R
Sandhoff
K
K
Proia
R L
RL
D000345:Affinity Labels; D000818:Animals; D019556:COS Cells; D002384:Catalysis; D002846:Chromatography, Affinity; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000345:Affinity Labels; D011994:Recombinant Proteins; D018698:Glutamic Acid; D001619:beta-N-Acetylhexosaminidases