Human adenylosuccinate lyase deficiency is an autosomal recessive disorder which results in autistic features, muscle wasting, and various magnitudes of mental retardation. Most of these ASL-deficiency-associated mutations occur in the central helical region that seems to be important for the stability of the tetramer (12
). In the present study we evaluated five disease-associated point mutations (R194C, K246E, R396C, R396H, and L311V) from various parts of the enzyme structure ( I).
For R194C mutant enzyme, the V at 25 °C, the rate constant for inactivation at 37 ° max C as well as the limiting activity and amount of tetramer at 37 °C are comparable to that of WT enzyme (Table ,). Furthermore, at 25 °C, the fluorescence intensity of R194C is comparable to native WT suggesting that overall conformation of R194C is comparable to native WT. When (A)R194 is mutated to Cys, the electrostatic attraction to (B)E464 is lost (-I). The crystal structure indicates that there is another Arg residue, (A)R234, which is even closer to (B)E464 (3.17 Å apart) that could be stabilizing the A/B and C/D subunit interfaces (-I). This (A)R234-(B)E464 interaction may be the reason why the R194C mutant enzyme is similar to WT enzyme at 25 °C and 37 °C. On the other hand, under harsh conditions such as 60 °C, the R194C mutant enzyme is the least stable enzyme. Preliminary data suggest that inclusion of the non-cleavable substrate analog, APBADP, in the incubation solution of R194C mutant enzyme at 60 °C provides appreciable stabilization of the enzyme. Thus, an appropriate nucleotide analog, by overcoming the instability of a disease-associated ASL mutant, may be useful in treating some types of ASL deficiency.
In contrast to R194C, the K246E mutant enzyme exhibits an extremely low Vmax
(only 2 % that of WT enzyme) and exists as a mixture of monomer (90 %) and aggregates at 37 °C. The K246E mutant enzyme, which is a charge reversal, exerts severe repulsion between (A)K246E and its partner, (B)D182 (-II). Furthermore, there are no other positively charged residues around (B)D182 to compensate for the absence of K246; hence a drastic destabilization of the human ASL tetramer is observed, yielding dimers or monomers. Since each catalytic site requires contributions of amino acid side chains from 3 subunits (6
), it is clear why the K246E enzyme is so low in activity.
Residue L311 is in the central helical region towards the middle ( I). At 25 °C, its Vmax
is ~76 % that of WT enzyme and the amount of tetramer is ~60 %, which is lower than that of WT enzyme (~80 %), while at 37 °C the amount of tetramer is similar to that of WT enzyme (~68 %). Since Val is less hydrophobic than Leu (27
), it might be expected that there would be weakening of the long range hydrophobic interactions in the L311V enzyme (27
). Replacing Leu by Val (with its lower molar volume) increases the flexibility of the hydration shell (28
) which leads to some destabilization of the tetramer, perhaps decreasing somewhat the subunit interactions as reflected in the reduced Hill coefficient (n = 1.3). In addition, its decreased fluorescence intensity indicates that the L311V enzyme has an altered conformation which can account for its diminished catalytic activity.
Our experimental results show that R396C and R396H mutant enzymes exhibit substantially decreased residual activity (~17 % that of WT enzyme) at 25 °C and 37 °C while maintaining similar amounts of tetramer as WT enzyme. Residue R396 is in close proximity to the entrance to the active site ( I), where its side chain points towards the solvent. Many proteins have positively charged Arg residues around the active site entrance while the side chain faces toward bulk water (30
). Water forms more ordered hydration shells around polar groups than the non-polar groups, leading to longer residence times of water (30
). This ordering of water extends the proteins’ electrostatic surfaces well away from their physical surfaces (made up of amino acid side chains) and increases the capturing of visiting ligands (30
), in this case the negatively charged SAMP or SAICAR. Any substitution for these positively charged Arg residues on the surface decreases the extended electrostatic surface, leading to diminished capturing of the substrate molecules and a decrease in activity, as in the R396H and R396C enzymes.
For wild type enzyme, the dependence of initial velocity on adenylosuccinate concentration provides the first evidence for kinetic cooperativity in this enzyme, probably because the kinetics were examined over a wider range of substrate concentration than previously. Also, we recently reported that direct binding of the non-cleavable substrate analog, APBADP, by human ASL shows positive cooperativity (7
); we now conclude that human ASL shows positive cooperativity in kinetics as well as in substrate binding. The K0.5
data of these disease-associated point mutations of ASL indicate that there are only small changes in the affinity of the enzyme for its substrate whereas the greatest change is in the extent of cooperativity. These changes in the extent of cooperativity might be due to the conformational changes caused by the disease-associated point mutations that lead to a change in the degree of communication among the subunits.
Such conformational changes were investigated using tryptophan fluorescence spectroscopy, which is extremely sensitive to any changes in the molecular environment and polarity (25
). In native
human ASL there are five Trp in each subunit: (B)W45 is surface exposed; (B) W39, (B) W43 and (B) W175 are buried; and (B) W326 is shared between B/A and B/C subunit interfaces ( II). However, the fluorescence emission intensity of native
WT enzyme at 25 °C is much greater than that of free Trp and WT enzyme in the presence of 6 M Gdn.HCl at 25 °C, implying that the local environment (within 6 A) of Trp is predominantly non-polar.
The fluorescence intensity of R194C mutant enzyme at 25 °C is similar to that of WT enzyme indicating that the overall conformation of R194C mutant enzyme is comparable to that of WT enzyme. The two human ASL crystal structures indicate that there are subtle changes near Arg194
during catalysis; therefore, changing Arg at position 194 to a Cys leads to a slight destabilization of the subunit interface without changing either Vmax
or the overall conformation, which reduces positive cooperativity as indicated by the Hill coefficient (n=1.3). A similar scenario has been documented for phenylalanine ammonia-lyase (34
In contrast, K246E mutant enzyme (which exists as a mixture of monomer-dimer along with some aggregates) has the lowest fluorescence intensity and is similar to that of the unfolded WT enzyme at 25 °C. These observations suggest overall conformational changes in the mutant protein greatly influence the communication among the subunits as indicated by the plot of initial velocity vs [SAMP] of K246E mutant enzyme, which exhibits apparent negative cooperativity (i.e., n is <1). It is possible that one site of K246E mutant enzyme binds substrate with high affinity and decreases the binding affinity for the enzyme’s other sites (35
). Alternatively, the apparent negative cooperativity can be attributed to the presence in the preparation of several species of active enzyme with different affinities for substrate (35
). For K246E mutant enzyme, the gel filtration data demonstrate that there are several species of the enzyme with different affinities for its substrate, so in this case that is probably the preferred explanation for the apparent negative cooperativity.
At 25 °C, as indicated by the fluorescence data the overall conformation of R396H mutant enzyme is different from native WT, whereas the overall conformation of R396C mutant enzyme is similar to that of native WT enzyme. According to the crystal structure, (A)R396 belongs to helix-19, where several residues in that helix have direct electrostatic interactions with the residues in subunits B and C and there is indirect stabilization of some of these inter-subunit interactions by (A)R396. Furthermore, a comparison between the two human ASL crystal structures (PDB # 2J91 and 2VD6) indicates that there are significant conformational changes in helix-19 after the catalysis of SAMP, which might be important for maintaining positive cooperativity. Changes in helix-19 may therefore cause destabilization of the tetramer and loss of inter-subunit communication, as indicated by the Hill coefficient (n = 1).
Most of the ASL deficient patients identified to date are compound heterozygotes (12
). The mutations that we investigated in the present study are found paired in patients as follows: R396H/L311V (37
) and R396C/R194C (9
). These two patients have been described as having severe mental retardation while the two reported patients with the K246E mutation exhibit moderate to severe mental retardation (38
). Although, the individual R194C and L311V mutant enzymes have comparable activity to WT enzyme, the genetic combination of R194C/R396C and L311V/R396H result in marked ASL deficiency. One or more of the following may explain the symptoms of these patients: 1. the protein might be a collection of hybrids (i.e., each tetramer is composed of some subunits of L311V and some of R396H) with kinetic and physicochemical characteristics different from those of either mutant alone or of the average of the two mutants; or 2. the expression of the mutant enzymes may be low. These possibilities may lead to a drastic decrease in catalytic activity, with resultant accumulation of S-Ado and SAICAriboside (the dephosphorylated substrates of SAMP and SAICAR) in high concentrations in the cerebrospinal fluid. These possibilities are currently being evaluated in this laboratory. It has been suggested that the accumulating S-Ado and/or SAICAriboside are toxic and may interact with the adenosine receptor in neurological tissues; however, experimental studies have not yet supported this possibility (13
). Although the biochemical mechanism of central nervous system damage has not been established, there is a clear association of adenylosuccinate lyase deficiency with autistic features and mental retardation. It is anticipated that elucidation of molecular basis of ASL deficiency may lead to effective pharmacological treatment of the disease.
This paper describes the study of five ASL-deficiency associated point mutations: K246E, R194C, L311V, R396C and R396H. We conclude that the most detrimental point mutation is the interfacial K246E mutant enzyme, which is observed as a monomer or dimer, but no tetramer, and has a residual activity of only 2% that of WT enzyme with no positive cooperativity. In contrast, the interfacial R194C mutant enzyme is very similar to WT enzyme in kinetic properties, and state of oligomerization at 25 °C and 37 °C; however, it exhibits marked thermal instability, which is best observed at 60 °C. The L311V mutation (located in the central helical region) results in an enzyme with a Vmax of 72% that of WT enzyme; however its state of oligomerization at 25 °C and 37 °C is similar to WT and shows conformational changes along with decreased positive cooperativity. Finally, the two replacements for Arg396, R396C and R396H, yield enzyme with the relatively low activity of ~17% that of WT, which exhibit conformational changes and loss of positive kinetic cooperativity; these effects are probably due to the location of Arg396 at the entrance to the active site. Thus, these disease-associated single mutations can yield enzymes with reduced activity either by affecting the catalytic reaction or by perturbing the enzyme’s multimeric structure and/or native conformation, which are required for catalytic function.