Role of βArg²¹¹ in the Active Site of Human β-Hexosaminidase B

Abstract

Tay-Sachs or Sandhoff disease results from a deficiency of either the alpha- or the beta-subunits of beta-hexosaminidase A, respectively. These evolutionarily related subunits have been grouped with the "Family 20" glycosidases. Molecular modeling of human hexosaminidase has been carried out on the basis of the three-dimensional structure of a bacterial member of Family 20, Serratia marcescens chitobiase. The primary sequence identity between the two enzymes is only 26% and restricted to their active site regions; therefore, the validity of this model must be determined experimentally. Because human hexosaminidase cannot be functionally expressed in bacteria, characterization of mutagenized hexosaminidase must be carried out using eukaryotic cell expression systems that all produce endogenous hexosaminidase activity. Even small amounts of endogenous enzyme can interfere with accurate K(m) or V(max) determinations. We report the expression, purification, and characterization of a C-terminal His(6)-tag precursor form of hexosaminidase B that is 99.99% free of endogenous enzyme from the host cells. Control experiments are reported confirming that the kinetic parameters of the His(6)-tag precursor are the same as the untagged precursor, which in turn are identical to the mature isoenzyme. Using highly purified wild-type and Arg(211)Lys-substituted hexosaminidase B, we reexamine the role of Arg(211) in the active site. As we previously reported, this very conservative substitution nevertheless reduces k(cat) by 500-fold. However, the removal of all endogenous activity has now allowed us to detect a 10-fold increase in K(m) that was not apparent in our previous study. That this increase in K(m) reflects a decrease in the strength of substrate binding was confirmed by the inability of the mutant isozyme to efficiently bind an immobilized substrate analogue, i.e., a hexosaminidase affinity column. Thus, Arg(211) is involved in substrate binding, as predicted by the chitobiase model, as well as catalysis.

Crystallographic Evidence for Substrate-assisted Catalysis in a Bacterial β-Hexosaminidase

“…This domain is homologous to domain III in SmCHB, and a structure-based sequence alignment demonstrated there to be a 29.5% sequence identity between the two domains, where 236 of the C ␣ atoms had a rms difference of 1.30 Å. What may indeed be a common feature of this (␤/␣) 8 barrel domain in family 20 glycosyl hydrolases is the conspicuous absence of regular helices at positions ␣5 and ␣7 in the (␤/␣) 8 barrel. In both SpHEX and SmCHB helix ␣5 consists of only a single turn of a 3/10 helix, whereas helix ␣7 is completely absent and is instead replaced by an extended loop.…”

“…Domain II of SpHEX is composed of residues 151-512 and is folded into a (␤/␣) 8 barrel with the active site of the enzyme residing at the C termini of the 8 ␤-strands of the barrel. This domain is homologous to domain III in SmCHB, and a structure-based sequence alignment demonstrated there to be a 29.5% sequence identity between the two domains, where 236 of the C ␣ atoms had a rms difference of 1.30 Å.…”

“…Overall, this domain in SpHEX contains shorter surface loops and is much more compact than its homologous counterpart, domain III, in SmCHB. Multiple sequence alignments of family 20 glycosyl hydrolases suggest that such a compact (␤/␣) 8 barrel domain may be a common feature among many family 20 ␤-hexosaminidases, including the human isoforms (10, 11).…”

“…Unlike the basic (␤/␣) 8 barrel motif, domain II of SpHEX contains three major loop structures that extend from the C termini of three of the 8 ␤-strands of the barrel. First, loop Lp7 replaces helix ␣7 as described above (Fig.…”

“…A substantial amount of genetic and biochemical information is available for these isozymes (5), but detailed information about their catalytic mechanism is limited. Mechanistic studies have been primarily limited by the difficulties in producing sufficient amounts of recombinant enzyme needed for kinetic analysis (6,7); however, recent improvements in expression and purification procedures have allowed more accurate kinetic measurements to be made (8). Crystals of human HexB have been grown (9); however, attempts at solving its three-dimensional structure have not been successful.…”