Active arginine residues in beta-hexosaminidase. Identification through studies of the B1 variant of Tay-Sachs disease.
Lysosomal beta-hexosaminidase (EC 3.2.1.52) occurs as two major isoenzymes, hexosaminidases A (alpha beta) and B (beta beta). The alpha- and beta-subunits are encoded by the HEXA and HEXB genes, respectively. Extensive homology in both the gene structures and deduced primary sequences demonstrate their common evolutionary origin. Defects in the alpha- or beta-subunits lead to Tay-Sachs of Sandhoff disease, respectively. The B1 variant of Tay-Sachs disease is characterized by an unusual phenotype. Patient samples contain both isoenzymes; however, hexosaminidase A lacks activity toward alpha-specific substrates. In a previous report, we analyzed the biochemical consequences of an Arg178----His substitution in the alpha-subunit, causing the B1 phenotype, by in vitro mutagenesis of the homologous codon for Arg211 in the beta-subunit to produce His. We found that the substitution did not affect dimer formation or cellular targeting but caused a near total loss of activity toward a common alpha- and/or beta-substrate. Additional effects were also noted that suggested a perturbation had occurred to the protein's secondary structure. In this report, we investigate further the role of Arg in the catalysis of hexosaminidase substrates. The introduction of more or less conservative amino acid substitutions at the beta-Arg211 site were evaluated in terms of their effects on the protein's catalytic activity and susceptibility to the arginine-specific reagents and on its stability and rate of maturation in the cell's lysosome. These data demonstrate that the changes in the in vivo stability and rate of maturation, previously noted with the Arg211----His substitution, are independent of the loss in enzymatic activity. Whereas treatment of purified normal human placental hexosaminidases A and B with arginine-specific modifying reagents produced a time-dependent loss of enzymatic activity toward both alpha-specific and common substrates, these reagents failed to significantly decrease the residual activities of mutant proteins lacking Arg at position 211. Kinetic analysis of the residual enzyme activity from our most conservative construct, Arg211----Lys, determined an apparent Vmax approximately 400-fold reduced from that of the wild type enzyme but detected no change in the apparent Km. Additionally, the pH optimum of this mutant enzyme was narrower and slightly more basic than that of the normal enzyme. Thus, Arg211 in the beta-subunit and, by extrapolation, the Arg178 in the alpha-subunit of beta-hexosaminidase are "active" residues, i.e. part of the catalytic sites, but do not participate in substrate binding.
Brown
C A
CA
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Mahuran
D J
DJ
D001120:Arginine / Q000737:chemistry*; D001483:Base Sequence; D015153:Blotting, Western; D002384:Catalysis; D002460:Cell Line; D003931:Diacetyl / Q000494:pharmacology; D054818:Hexosaminidase A; D054819:Hexosaminidase B; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics*; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation; D010658:Phenylglyoxal / Q000494:pharmacology; D013661:Tay-Sachs Disease / Q000235:genetics*; D014162:Transfection; D001619:beta-N-Acetylhexosaminidases / Q000037:antagonists & inhibitors / Q000235:genetics*
D007527:Isoenzymes; D001120:Arginine; D054818:Hexosaminidase A; D054819:Hexosaminidase B; D001619:beta-N-Acetylhexosaminidases; D003931:Diacetyl; D010658:Phenylglyoxal