Conserved Residues in the Subunit Interface of tau Glutathione S‐transferase Affect Catalytic and Structural Functions

The tau class glutathione S‐transferases (GSTs) have important roles in stress tolerance and the detoxification of herbicides in crops and weeds. Structural investigations of a wheat tau GST (TaGSTU4) show two subunit interactions: a hydrogen bond between the Tyr93 and Pro65 from another subunit of the dimer, and two salt bridges between residues Glu78 and side chains of Arg95 and Arg99 in the opposite subunit. By investigating enzyme activities, kinetic parameters and structural characterizations, this study showed the following results: (i) the hydrogen bond interaction between the Tyr93 and Pro65 was not essential for dimerization, but contributed to the enzyme's catalytic activity, thermal stability and affinity towards substrates glutathione and 1‐chloro‐2, 4‐dinitrobenzene; and (ii) two salt bridges mainly contributed to the protein structure stability and catalysis. The results of this study form a structural and functional basis for rational design of more selective and environmentally friendly herbicides.     

Detailed mechanisms of the inactivation of factor VIIIa by activated protein C in the presence of its cofactors, protein S and factor V

Factor VIIIa is inactivated by a combination of two mechanisms. Activation of factor VIII by thrombin results in a heterotrimeric factor VIIIa that spontaneously inactivates due to dissociation of the A2 subunit. Additionally, factor VIIIa is cleaved by the anticoagulant serine protease, activated protein C, at two cleavage sites, Arg(336) in the A1 subunit and Arg(562) in the A2 subunit. We previously characterized an engineered variant of factor VIII which contains a disulfide bond between the A2 and the A3 subunits that prevents the spontaneous dissociation of the A2 subunit following thrombin activation. Thus, in the absence of activated protein C, this variant has stable activity following activation by thrombin. To isolate the effects of the individual activated protein C cleavage sites on factor VIIIa, we engineered mutations of the activated protein C cleavage sites into the disulfide bond-cross-linked factor VIII variant. Arg(336) cleavage is 6-fold faster than Arg(562) cleavage, and the Arg(336) cleavage does not fully inactivate factor VIIIa when A2 subunit dissociation is blocked. Protein S enhances both cleavage rates but enhances Arg(562) cleavage more than Arg(336) cleavage. Factor V also enhances both cleavage rates when protein S is present. Factor V enhances Arg(562) cleavage more than Arg(336) cleavage as well. As a result, in the presence of both activated protein C cofactors, Arg(336) cleavage is only twice as fast as Arg(562) cleavage. Therefore, both cleavages contribute significantly to factor VIIIa inactivation.     

Proteolysis of non-phosphorylated and phosphorylated tau by thrombin

The microtubule-associated protein tau aggregates intracellularly by unknown mechanisms in Alzheimer's disease and other tauopathies. A contributing factor may be a failure to break down free cytosolic tau, thus creating a surplus for aggregation, although the proteases that degrade tau in brain remain unknown. To address this issue, we prepared cytosolic fractions from five normal human brains and from perfused rat brains and incubated them with or without protease inhibitors. D-Phenylalanyl-L-prolylarginyl chloromethyl ketone, a thrombin-specific inhibitor, prevented tau breakdown in these fractions, suggesting that thrombin is a brain protease that processes tau. We next exposed human recombinant tau to purified human thrombin and analyzed the fragments by N-terminal sequencing. We found that thrombin proteolyzed tau at multiple arginine and lysine sites. These include Arg(155)-Gly(156), Arg(209)-Ser(210), Arg(230)-Thr(231), Lys(257)-Ser(258), and Lys(340)-Ser(341) (numbering according to the longest human tau isoform). Temporally, the initial cleavage occurred at the Arg(155)-Gly(156) bond. Proteolysis of the resultant C-terminal tau fragment then proceeded bidirectionally. When tau was phosphorylated by glycogen synthase kinase-3beta, most of these proteolytic processes were inhibited, except for the first cleavage at the Arg(155)-Gly(156) bond. Furthermore, paired helical filament tau prepared from Alzheimer's disease brain was more resistant to thrombin proteolysis than following dephosphorylation by alkaline phosphatase. The results suggest a possible role for thrombin in proteolysis of tau under physiological and/or pathological conditions in human brains. They are consistent with the hypothesis that phosphorylation of tau inhibits proteolysis by thrombin or other endogenous proteases, leading to aggregation of tau into insoluble fibrils.     

Tau peptides and tau mutant protein aggregation inhibition by cationic polyethyleneimine and polyarginine

Tau protein plays a major role in Alzheimer's disease. The tau protein loses its functionality by self‐aggregation due to the two six‐amino acid sequences VQIVYK and VQIINK of the protein. Hence it is imperative to find therapeutics that could inhibit the self‐aggregation of this tau peptide fragments. Here, we study the inhibitory potential of a cationic polymer polyethyleneimine (PEI) and a cationic polypeptide arginine (Arg) on the aggregation of VQIVYK, and GKVQIINKLDL peptides, and tau mutant protein (P301L), found frequently in taupathy. Various characterization methods are employed including thioflavin S, transmission electron microscopy, and dynamic light scattering to study the aggregation/inhibition process in vitro. Results show that PEI and Arg significantly inhibit tau peptides and protein aggregation. The study could be applied to understand tau protein aggregation mechanism in the presence of cationic polymers.     

Interactions between the microtubule-associated tau proteins and S100b regulate tau phosphorylation by the Ca2+/calmodulin-dependent protein kinase II

Binding between the microtubule-associated tau protein and S100b protein was demonstrated by affinity chromatography and cross-linking experiments and was manifested in the effect of S100b on tau protein phosphorylation by protein kinase II. All three expressions of the binding showed that S100b discriminates among the four species of tau, revealing for the first time that the different kinds of tau may differ functionally. Noncovalent interaction between tau and S100b depended on the presence of Ca2+ or Zn2+ and resulted in total inhibition of tau phosphorylation by protein kinase II. In the absence of reducing agent, covalent binding studies between Cys84 beta in the carboxyl-terminal region of the S100b-beta subunit and tau proteins confirmed interactions between the two proteins. It is suggested that the homologous calcium-binding domain that characterizes the carboxyl terminus of S100 and the tubulin subunit may be responsible for the common interaction of both proteins with tau proteins. The physicochemical relationship between S100 subunits and p11, the subunit of a substrate for tyrosine kinase, and their similarity in interaction with protein kinase substrates are discussed.     

Identification of the allosteric regulatory site in bacterial phosphoribulokinase

Bacterial phosphoribulokinases (PRKs) are octameric members of the adenylate kinase family of enzymes. The enzyme is allosterically activated by NADH and allosterically inhibited by AMP. We have determined the crystal structure of PRK from Rhodobacter sphaeroides bound to the ATP analogue AMP-PCP to a resolution of 2.6 A. The structure reveals that the ATP analogue does not bind to the canonical ATP site found in adenylate kinase family members. Rather, the AMP-PCP binds in two different orientations at the interface of three of the monomers in the octamer. This interface was previously characterized as having an unusually large number of arginine residues. Of the five arginine residues that are near the bound nucleotide, one (Arg 221) is highly conserved in both prokaryotic and eukaryotic (nonallosterically regulated) PRKs, two (Arg 234 and Arg 257) are on a second subunit and conserved in only prokaryotic PRKs, and two (Arg 30 and Arg 31) are on a third subunit with only one of them (Arg 31) conserved in prokaryotic PRKs. Each of these arginine residues was converted by site-directed mutagenesis to alanine. Fluorescence binding data suggest that none of these arginines are involved in active site ATP binding and that Arg 234 and Arg 257 on the second subunit are directly involved in NADH binding, while the other arginines have a minimal effect on NADH binding. While the wild-type enzyme exhibits low maximal activity and hyperbolic kinetics with respect to ATP in the absence of NADH and high maximal activity and sigmoidal kinetics in the presence of NADH, the R31A mutant exhibits identical hyperbolic kinetics with respect to ATP in the presence or absence of NADH. Thus, the transmission of allosteric information from one subunit to another is conducted through a single path that includes NADH and Arg 31.     

Constitution of the twin polymerase of DNA polymerase III holoenzyme

It is speculated that DNA polymerases which duplicate chromosomes are dimeric to provide concurrent replication of both leading and lagging strands. DNA polymerase III holoenzyme (holoenzyme), is the 10-subunit replicase of the Escherichia coli chromosome. A complex of the alpha (DNA polymerase) and epsilon (3'-5' exonuclease) subunits of the holoenzyme contains only one of each protein. Presumably, one of the eight other subunit(s) functions to dimerize the alpha epsilon polymerase within the holoenzyme. Based on dimeric subassemblies of the holoenzyme, two subunits have been elected as possible agents of polymerase dimerization, one of which is the tau subunit (McHenry, C. S. (1982) J. Biol. Chem. 257, 2657-2663). Here, we have used pure alpha, epsilon, and tau subunits in binding studies to determine whether tau can dimerize the polymerase. We find tau binds directly to alpha. Whereas alpha is monomeric, tau is a dimer in its native state and thereby serves as an efficient scaffold to dimerize the polymerase. The epsilon subunit does not associate directly with tau but becomes dimerized in the alpha epsilon tau complex by virtue of its interaction with alpha. We have analyzed the dimeric alpha epsilon tau complex by different physical methods to increase the confidence that this complex truly contains a dimeric polymerase. The tau subunit is comprised of the NH2-terminal two-thirds of tau but does not bind to alpha epsilon, identifying the COOH-terminal region of tau as essential to its polymerase dimerization function. The significance of these results with respect to the organization of subunits within the holoenzyme is discussed.     

Calmodulin activates intersubunit electron transfer in the neuronal nitric-oxide synthase dimer

Neuronal nitric oxide synthase (nNOS) is composed of an oxygenase domain that binds heme, (6R)-tetrahydrobiopterin, and Arg, coupled to a reductase domain that binds FAD, FMN, and NADPH. Activity requires dimeric interaction between two oxygenase domains and calmodulin binding between the reductase and oxygenase domains, which triggers electron transfer between flavin and heme groups. We constructed four different nNOS heterodimers to determine the path of calmodulin-induced electron transfer in a nNOS dimer. A predominantly monomeric mutant of rat nNOS (G671A) and its Arg binding mutant (G671A/E592A) were used as full-length subunits, along with oxygenase domain partners that either did or did not contain the E592A mutation. The E592A mutation prevented Arg binding to the oxygenase domain in which it was present. It also prevented NO synthesis when it was located in the oxygenase domain adjacent to the full-length subunit. However, it had no effect when present in the full-length subunit (i.e. the subunit containing the reductase domain). The active heterodimer (G671A/E592A full-length subunit plus wild type oxygenase domain subunit) showed remarkable similarity with wild type homodimeric nNOS in its catalytic responses to five different forms and chimeras of calmodulin. This reveals an active involvement of calmodulin in supporting transelectron transfer between flavin and heme groups on adjacent subunits in nNOS. In summary, we propose that calmodulin functions to properly align adjacent reductase and the oxygenase domains in a nNOS dimer for electron transfer between them, leading to NO synthesis by the heme.     

tau binds and organizes Escherichia coli replication through distinct domains. Partial proteolysis of terminally tagged tau to determine candidate domains and to assign domain V as the alpha binding domain

The tau subunit dimerizes Escherichia coli DNA polymerase III core through interactions with the alpha subunit. In addition to playing critical roles in the structural organization of the holoenzyme, tau mediates intersubunit communications required for efficient replication fork function. We identified potential structural domains of this multifunctional subunit by limited proteolysis of C-terminal biotin-tagged tau proteins. The cleavage sites of each of eight different proteases were found to be clustered within four regions of the tau subunit. The second susceptible region corresponds to the hinge between domain II and III of the highly homologous delta' subunit, and the third region is near the C-terminal end of the tau-delta' alignment (Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J. (1997) Cell 91, 335-345). We propose a five-domain structure for the tau protein. Domains I and II are based on the crystallographic structure of delta' by Guenther and colleagues. Domains III-V are based on our protease cleavage results. Using this information, we expressed biotin-tagged tau proteins lacking specific protease-resistant domains and analyzed their binding to the alpha subunit by surface plasmon resonance. Results from these studies indicated that the alpha binding site of tau lies within its C-terminal 147 residues (domain V).     

Communication between the two active sites of glutathione S-transferase A1-1, probed using wild-type-mutant heterodimers

To study the communication between the two active sites of dimeric glutathione S-transferase A1-1, we used heterodimers containing one wild-type (WT) active site and one active site with a single mutation at either Tyr9, Arg15, or Arg131. Tyr9 and Arg15 are part of the active site of the same subunit, while Arg131 contributes to the active site of the opposite subunit. The V(max) values of Tyr9 and Arg15 mutant enzymes were less than 2% that of WT, indicating their importance in catalysis. In contrast, V(max) values of Arg131 mutant enzymes were about 50-90% of that of WT enzyme while K(m)(GSH) values were approximately 3-8 times that of WT, suggesting that Arg131 plays a role in glutathione binding. The mutant enzyme (with a His(6) tag) and the WT enzyme (without a His(6) tag) were used to construct heterodimers (WT-Y9F, WT-Y9T, WT-R15Q, WT-R131M, WT-R131Q, and WT-R131E) by incubation of a mixture of wild-type and mutant enzyme at pH 7.5 in buffer containing 1,6-hexanediol, followed by dialysis against buffer lacking the organic solvent. The resultant heterodimers were separated from the wild-type and mutant homodimers using chromatography on nickel-nitrilotriacetic acid agarose. The V(max) values of all heterodimers were lower than expected for independent active sites. Our experiments demonstrate that mutation of an amino acid residue in one active site affects the activity in the other active site. Modeling studies show that key amino acid residues and water molecules connect the two active sites. This connectivity is responsible for the cross-talk between the active sites.     

DNA Polymerase III holoenzyme of Escherichia coli. IV. The holoenzyme is an asymmetric dimer with twin active sites

Pol III, a subassembly of Escherichia coli DNA polymerase III holoenzyme lacking only the auxiliary beta subunit, was purified to homogeneity by an improved procedure. This assembly consists of nine different polypeptides, likely in a 1:1 stoichiometry: a catalytic core (pol III) of alpha (132 kDa), epsilon (27 kDa), and theta (10 kDa), and six auxiliary subunits: tau (71 kDa), gamma (52 kDa), delta (35 kDa), delta' (33 kDa), chi (15 kDa), and psi (12 kDa). The assembly behaves on gel filtration as a particle of about 800 kDa, indicating a content of two each of the subunits. A new procedure for purifying the core yielded a novel dimeric form which may provide the foundation for the dimeric nature of the more complex pol III and holoenzyme forms. Pol III readily dissociates into several subassemblies including pol III', likely a dimeric core with two tau subunits. The holoenzyme, purified by a similar procedure with ATP and Mg2+ present throughout, retained the beta subunit (37 kDa) as well as all the subunits present in pol III; the mass of the holoenzyme was estimated to be 900 kDa. The isolated initiation complex of holoenzyme with a primed template DNA and the elongation complex (formed in the presence of three deoxynucleoside triphosphates) had the same composition and stoichiometry as observed for pol III with two beta dimers in addition. An initiation complex assembled from a mixture of monomeric pol III core, gamma 2 delta delta' chi psi complex (gamma complex), beta, and tau retained the core, one beta dimer, and two tau subunits but was deficient in the gamma complex. When tau was omitted from the assembly mixture, the initiation complex contained one or two gamma complexes instead of the tau subunit. Based on these data, pol III holoenzyme is judged to be an asymmetric dimeric particle with twin pol III core active sites and two different sets of auxiliary units designed to achieve essentially concurrent replication of both leading and lagging strand templates.     

Rearrangements in a hydrophobic core region mediate cAMP action in the regulatory subunit of PKA

cAMP-dependent protein kinase (PKA) forms an inactive heterotetramer of two regulatory (R; with two cAMP-binding domains A and B each) and two catalytic (C) subunits. Upon the binding of four cAMP molecules to the R dimer, the monomeric C subunits dissociate. Based on sequence analysis of cyclic nucleotide-binding domains in prokaryotes and eukaryotes and on crystal structures of cAMP-bound R subunit and cyclic nucleotide-free Epac (exchange protein directly activated by cAMP), four amino acids were identified (Leu203, Tyr229, Arg239 and Arg241) and probed for cAMP binding to the R subunits and for R/C interaction. Arg239 and Arg241 (mutated to Ala and Glu) displayed no differences in the parameters investigated. In contrast, Leu203 (mutated to Ala and Trp) and Tyr229 (mutated to Ala and Thr) exhibited up to 30-fold reduced binding affinity for the C subunit and up to 120-fold reduced binding affinity for cAMP. Tyr229Asp showed the most severe effects, with 350-fold reduced affinity for cAMP and no detectable binding to the C subunit. Based on these results and structural data in the cAMP-binding domain, a switch mechanism via a hydrophobic core region is postulated that is comparable to an activation model proposed for Epac.     

Pairwise electrostatic interactions between alpha-neurotoxins and gamma, delta, and epsilon subunits of the nicotinic acetylcholine receptor

alpha-Neurotoxins bind with high affinity to alpha-gamma and alpha-delta subunit interfaces of the nicotinic acetylcholine receptor. Since this high affinity complex likely involves a van der Waals surface area of approximately 1200 A(2) and 25-35 residues on the receptor surface, analysis of side chains should delineate major interactions and the orientation of bound alpha-neurotoxin. Three distinct regions on the gamma subunit, defined by Trp(55), Leu(119), Asp(174), and Glu(176), contribute to alpha-toxin affinity. Of six charge reversal mutations on the three loops of Naja mossambica mossambica alpha-toxin, Lys(27) --> Glu, Arg(33) --> Glu, and Arg(36) --> Glu in loop II reduce binding energy substantially, while mutations in loops I and III have little effect. Paired residues were analyzed by thermodynamic mutant cycles to delineate electrostatic linkages between the six alpha-toxin charge reversal mutations and three key residues on the gamma subunit. Large coupling energies were found between Arg(33) at the tip of loop II and gammaLeu(119) (-5.7 kcal/mol) and between Lys(27) and gammaGlu(176) (-5.9 kcal/mol). gammaTrp(55) couples strongly to both Arg(33) and Lys(27), whereas gammaAsp(174) couples minimally to charged alpha-toxin residues. Arg(36), despite strong energetic contributions, does not partner with any gamma subunit residues, perhaps indicating its proximity to the alpha subunit. By analyzing cationic, neutral and anionic residues in the mutant cycles, interactions at gamma176 and gamma119 can be distinguished from those at gamma55.     

Cross-Linking Sites of the Human Tau Protein, Probed by Reactions with Human Transglutaminase

Abstract: A portion of the neurofibrillary tangles of Alzheimer's disease has the characteristics of cross-linked protein. Because the principal component of these lesions is the microtubule-associated protein tau, and because a major source of cross-linking activity within neurons is supplied by tissue transglutaminase (TGase), it has been postulated that isopeptide bond formation is a major posttranslational modification leading to the formation of insoluble neurofibrillary tangles. Here we have mapped the sites on two isoforms of human tau protein (τ23 and τ40) capable of participating in human TGase-mediated isopeptide bond formation. Using dansyl-labeled fluorescent probes, it was shown that eight Gln residues can function as amine acceptor residues, with two major sites being Gln³⁵¹ and Gln⁴²⁴. In addition, 10 Lys residues were identified as amine donors, most of which are clustered adjacent to the microtubule-binding repeats of tau in regions known to be solvent accessible in filamentous tau. The distribution of amine donors correlated closely with that of Arg residues, suggesting a link between neighboring positive charge and the TGase selectivity for donor sites in the protein substrate. Apart from revealing the sites that can be cross-linked during the TGase-catalyzed assembly of tau filaments, the results suggest a topography for the tau monomers so assembled.     

DNA and RNA-DNA annealing activity associated with the tau subunit of the Escherichia coli DNA polymerase III holoenzyme.

The DNA polymerase III (pol III)holoenzyme is the 10 subunit replicase of Escherichia coli. The 71 kDa tau subunit, encoded by dnaX, dimerizes the core polymerase (alpha epsilon theta) to form pol III'[(alpha epsilon theta)2 tau 2]. tau is also a single-stranded DNA-dependent ATPase and can substitute for the gamma subunit during initiation complex formation. We show here that tau also possesses a DNA-DNA and RNA-DNA annealing activity that is stimulated by Mg2+, but neither requires ATP nor is inhibited by non-hydrolyzable ATP analogs. This suggests the tau may act to stabilize the primer-template interaction during DNA replication.     

ATP interactions of the tau and gamma subunits of DNA polymerase III holoenzyme of Escherichia coli

The tau and gamma subunits of the DNA polymerase III holoenzyme of Escherichia coli were each isolated in large quantities as oligomers from overproducing cells in which their genes (dnaZ and X) were under the control of a T7 phage promoter. The 52-kDa gamma subunit (encoded by the dnaZ sequence) contains three-forths of the N-terminal residues of the 71-kDa tau subunit (encoded by the dnaX sequence). Both gamma and tau share a binding site for ATP (or dATP). A DNA-dependent ATPase activity (Lee, S.H., and Walker, J.R. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2713-2717) exhibited only by the tau subunit, presumably requires a DNA-binding site in the C-terminal domain lacking in the gamma subunit. Among ATPases dependent on single-stranded DNA, the tau activity is remarkable in the failure of homopolymers (e.g. poly(dA) or poly(dT)) to replace natural DNAs. The presumed need for certain secondary structures may reflect a feature of template binding in the crucial contribution that tau makes to the high processivity of polymerase III holoenzyme. Limited tryptic digestion of tau generates a fragment that resembles gamma in: (i) size, (ii) binding of ATP without ATPase activity, and (iii) a level of complementing holoenzyme activity in extracts of dnaZ-mutant cells that is higher than that of tau.     

Relation of the Escherichia coli dnaX gene to its two products--the tau and gamma subunits of DNA polymerase III holoenzyme.

The Escherichia coli DNA polymerase III holoenzyme 71.1 kDa tau subunit is a 643 amino acid protein encoded by the dnaX gene. This gene also encodes the holoenzyme 56.5 kDa gamma subunit. The tau factor (as a tau'-LacZ' fusion protein) has been isolated and shown to be cleaved in vitro to form gamma and a 135 kda C-terminal cleavage product. The tau'-LacZ' fusion protein, gamma, and the C-terminal cleavage product have been isolated. N-terminal sequencing has demonstrated that tau and gamma share the same N-terminal sequences and that tau is proteolytically cleaved in vitro between residues 498 and 499 to form gamma. In addition, residues 420-440 were shown to be present in both tau and gamma by use of antibody specific for a synthetic peptide corresponding to that sequence. Some mechanism functions in vivo to ensure that tau and gamma are synthesized in a ratio of about one-to-one, as shown by radioimmune precipitation of tau and gamma from cellular extracts.     

Introduction of the alpha subunit mutation associated with the B1 variant of Tay-Sachs disease into the beta subunit produces a beta-hexosaminidase B without catalytic activity

Lysosomal beta-hexosaminidase (beta-N-acetylhexosaminidase, EC 3.2.1.52) occurs in two major isozyme forms, hexosaminidase A (alpha beta) and hexosaminidase B (beta beta). Although dimer formation is required for enzymatic activity, both subunits contain active sites which share many common substrates. However, the alpha subunit alone confers on hexosaminidase A the specificity for negatively charged substrates, e.g. GM2 ganglioside. Recently, a point mutation, producing a single amino acid substitution in the alpha subunit (Arg178-His), has been found to be associated with the B1 variant phenotype of Tay-Sachs disease (Ohno, K., and Suzuki, K. (1988) J. Neurochem. 50, 316-318). This variant is characterized by normal levels of hexosaminidase A as measured by a common artificial substrate, but an absence of activity toward alpha subunit-specific substrates. However, because of the presence of an active beta subunit in the mutant hexosaminidase A, it has not been possible to determine whether the affected alpha subunit has undergone a change in substrate specificity or become totally inactive. In order to define the full effect of the B1 mutation we have taken advantage of the common evolutionary origin of the genes coding for the alpha and beta subunits. Since the B1 mutation occurs in a region of extended identity between the two subunits, we have duplicated the Arg178-His mutation in a cDNA coding for the human beta subunit (Arg211-His). By expression of the mutant construct in monkey COS cells we have been able to examine the effect of this mutation on beta subunits which are capable of forming stable, active homodimers, an experiment that could not readily be accomplished with heterodimeric hexosaminidase A. Our data show that beta homodimers containing the Arg211-His substitution are formed and are transported into the lysosome in a manner identical to that of normal pro-hexosaminidase B. However, the mutant homodimers are processed at a slower rate and are less stable in the lysozyme. Their most striking feature was a total lack of normal hexosaminidase B activity. We conclude that while the effect of the Arg178-His substitution is not strictly limited to the active site, the severe B1 phenotype results from a totally inactive alpha-subunit in hexosaminidase A.