The discovery of a novel activator of ALDH2*1 and ALDH2*2 (ref. 3
), and the desire to improve the properties of this interesting lead compound led us to characterize the mechanism of activation of Alda-1. First, we sought structural insight into the interaction of Alda-1 with ALDH2. The structure of the binary complexes between Alda-1 and ALDH2*1 and ALDH2*2 were solved by X-ray crystallography. The site to which Alda-1 binds was surprising and generated several working hypotheses for how ALDH2*1 and ALDH2*2 are activated. With this new information in mind, we designed a series of kinetics experiments to characterize the substrate and reaction specificity of activation.
The location of Alda-1 binding within the substrate entrance tunnel of ALDH2 is reminiscent of the binding of daidzin, a known potent inhibitor of ALDH2 (ref. 13
). If the positions of Alda-1 and daidzin in their respective crystal structures are correct, we reasoned that Alda-1 should antagonize daidzin inhibition. As shown in and (see also ref. 3
), we found this to be true for both the wild-type, ALDH2*1, and for the mutant, ALDH2*2, confirming that Alda-1 and daidzin share overlapping binding sites. The very different effects of daidzin and Alda-1 on ALDH2 activity can be explained, in part, from their crystal structures. In the daidzin bound structure13
, the phenolic moiety interacts directly with two essential active site residues, Cys302 and Glu268, inhibiting the enzyme by restricting substrate binding and catalysis. In contrast, our structure shows that Alda-1 binds at the entrance to the active site, but does not sterically interfere with the catalytic residues.
Because Alda-1 does block part of the substrate site, we predicted that ALDH2 activation would depend on substrate size. Modeling of the complex suggests that the space between Cys302 and the benzodioxol ring of Alda-1 could accommodate acyl-enzyme intermediates up to 4 carbons in length (). Hence, we examined the concentration dependence of Alda-1 activation at saturating concentrations of acetaldehyde, propionaldehyde, butyraldehyde, benzaldehyde, phenylacetaldehyde, and 4-trans-(N,N-dimethylamino)-cinnamaldehyde (DACA). We found that only the smaller linear aliphatic aldehydes were activated by Alda-1 and the extent of activation decreased with length (). Alda-1 had little effect on activity with benzaldehyde, even at the maximum concentration used; 200 μM. Alda-1 also had little effect on ALDH2 activity with phenylaceteldehyde or DACA, although high concentrations of Alda-1 (>100 μM) were weakly inhibitory. Of course, these data do not explain how substrate is able to gain access to the active site when Alda-1 is bound in the substrate entrance tunnel, nor do they shed light on why Alda-1 is an activator rather than an inhibitor of ALDH2.
Model of the potential mechanism for activation of ALDH2 by Alda-1
The answers to these questions reside in the unique nature of coenzyme binding to ALDHs. In all the ALDH family members, the active site and the NAD+
coenzyme binding site are connected, forming a tunnel through the enzyme. One rationale for the ordered binding of coenzyme prior to that of aldehyde substrate is that the binding of coenzyme helps to retain the substrate within the active site by lowering the frequency of its exit from the coenzyme-binding site side of this tunnel. shows a hypothetical quaternary complex of ALDH2, an acyl-enzyme intermediate of butyraldehyde, NAD+
, and Alda-1. Although Alda-1 blocks the substrate entrance tunnel, we propose that substrate access and product release can still occur - through the nicotinamide cleft. This is possible because of the well-characterized flexibility of the nicotinamide mononucleotide moiety in ALDH2 (refs. 14,15
). We and others have demonstrated that the nicotinamide half of the NAD+
coenzyme retains substantial flexibility when bound to ALDH isozymes. In ALDH2, there appear to be two relatively stable conformations relevant to catalysis; with the nicotinamide ring adjacent to or rotated away from Cys302 (ref. 15
). We also know from NMR studies that both NAD+
and NADH are rapidly sampling a number of conformations when bound to ALDH2 (ref. 14
). In addition, several alternate cofactor conformations have been observed in crystal structures of other ALDH family members16–18
. Hence, the coenzyme can - and probably does - sample multiple conformations during catalysis which permit both substrate binding and product release even when Alda-1 is bound.
Activation of dehydrogenase activity by Alda-1 is most likely achieved through acceleration of acyl-enzyme hydrolysis, the rate-limiting step in ALDH2*1. Clearly, limiting the substrate access to the nicotinamide cleft could slow the rate of substrate binding or product release. However, the Vmax
values for aldehyde oxidation by ALDH2*1 are close to diffusion-limited conditions, so even a substantial decrease in aldehyde association rates will not be observed in steady state measurements, where the rates of catalysis are approximately five orders of magnitude lower. On the other hand, we know from the effects of Alda-1 and NAD+
on the esterase reaction that the simultaneous presence of both molecules accelerates ester hydrolysis, even though the rate-limiting step, in the absence of activators, is known to be acylation19,20
. Consequently, access through the nicotinamide cleft may not appreciably slow substrate access. The fact that Alda-1 activates ALDH2*1 approximately 2-fold for the dehydrogenase reaction suggests that the activated water molecule, on average, has half as many non-productive encounters within the active site. Thus, we propose that by binding at and blocking one of the exits from the active site, Alda-1 increases the likelihood of a productive encounter between the activated water molecule and the thioacyl intermediate (), moderately accelerating acyl-enzyme hydrolysis in ALDH2*1.
Another important feature of Alda-1 is its ability to protect ALDH2 from inactivation due to adduct formation between the substrate and key Cys residues in the enzyme. Reduction in ALDH activity occurs in a number of disease states associated with oxidative stress21,22
. We previously reported that in the presence of 100 μM of 4-hydroxynonenal, ALDH2 activity is abolished within a couple of minutes3
. However, 20 μM Alda-1 prevented 4HNE-induced ALDH2 inactivation. It appears likely that Alda-1 reduces accessibility of Cys302, as well as the adjacent Cys301 and Cys303 residues, in the catalytic channel to 4-HNE adduct formation and the resulting enzyme inactivation3
It is interesting that both coenzyme and Alda-1 are nonessential activators for the esterase reaction in ALDH2. Neither coenzyme nor Alda-1 participates in the chemical events, but the presence of either activates catalysis in a saturable manner. The ability of NAD+
and NADH to increase the rate of ester hydrolysis in ALDH2 isozymes is well-established and it is generally thought that this occurs through an increase in the nucleophilicity of Cys30217
. Indeed, there is evidence that coenzyme binding is associated with a decrease in the pKa
of the active site nucleophile in ALDH family members and provides a chemical and kinetic rationale for the ordered binding of coenzyme prior to the substrate aldehyde in the dehydrogenation reaction23
. We show here that Alda-1 induces a similar increase in reactivity towards esters (), but this does not appear to be associated with an appreciable shift in the pKa
of Cys302 upon Alda-1 binding (apparent pKa
values of 7.8 and 7.6, respectively). Thus, it is likely that esterase activation occurs primarily by preventing the ester from non-productively transiting the active site through the substrate binding tunnel (). This is consistent with our proposed mechanism of dehydrogenase activation, where blocking one exit would effectively increase the number of productive encounters with Cys302. This mechanism could also be a component of esterase activation by coenzyme; whether coenzyme is bound in the hydride transfer or hydrolysis positions15
, it would reduce the chances of non-productive escape through the coenzyme binding cleft.
Modeled complex between ALDH2, Alda-1 and para-nitrophenylacetate (pNPA)
The fact that the actions of coenzyme and Alda-1 are additive suggests that the presence of both activators does not restrict access to Cys302 enough to slow the rate limiting acylation step for ester hydrolysis19,20
. Thus, we propose that nonessential activator binding at either end of the substrate-to-coenzyme tunnel in ALDH would limit diffusion of the substrate straight through the enzyme and thereby increase the effective concentration of the reacting groups within the active site. Like the dehydrogenase reaction, we predict that activation of the esterase reaction by Alda-1 will depend on the size of the ester; the leaving group will need to fit within the nicotinamide cleft and the acylating portion will likely be limited to four or fewer carbons (). A dependence of the rate of ester hydrolysis on the size of the acylating portion has been reported and is in agreement with this prediction24
. Here again, the conformational flexibility of the nicotinamide moiety is essential to facilitate both the binding of the ester and release of the products through the nicotinamide cleft.
Consistent with the structural information gleaned from the complex between Alda-1 and ALDH2*2, Alda-1 partially restores both the kcat
values for NAD+
in ALDH2*2 (). We also show that, in the presence of both coenzyme and Alda-1, esterase activity is increased to a greater extent in ALDH2*2 than in wild-type ALDH2 (100-fold vs 10-fold, respectively; ). Together, these results suggest that Alda-1 binding to ALDH2*2 does more than increase the effective concentration of substrates within the active site; namely that Alda-1 directly promotes the structural and functional rescue of ALDH2*2 (). Our lab has previously shown that the exchange of Lys for Glu487 in ALDH2*2 results in the loss of structural integrity to the coenzyme-binding and active sites9,11
. This disordering gives rise to the 200-fold elevation in KM
and the 10-fold reduction in maximal velocity10
. In general, the disorder of the coenzyme binding site begins at residue 246 and continues through Glu268 and the catalytic site is disordered in the immediate vicinity of Glu268, beginning at Phe465 and continuing on through Glu478 (ref. 9
). Even when the structure of the coenzyme-binding site is restored upon coenzyme binding, the regions surrounding the coenzyme and active sites remain less ordered than in the wild-type enzyme11
. Two of the active site residues that remain disordered are Glu268, the general base for catalysis, and Glu399, which stabilizes the position of the nicotinamide ring. Alda-1 does not directly contact any of the aforementioned residues, but does form close interactions with Phe459 and Trp177, both of which are in immediate proximity to Phe465 and Glu268. Thus, we hypothesize that through these interactions, binding of Alda-1 could redirect the aberrant dynamics in both regions of the enzyme to improve catalytic efficiency. Similarly, Alda-1 increases the maximal velocity of ALDH2*1 by reducing the number of non-productive encounters between the reacting groups in the active site and thereby accelerates catalysis. Thus, Alda-1 represents a new pharmacological agonist: it increases productive substrate-enzyme interaction and protects the enzyme from substrate-induced inactivation. Further, Alda-1 acts as a chemical chaperone by stabilizing the structurally-impaired enzyme at the tetrameric interface as well as within the catalytic tunnel, leading to catalytic recovery. This is a variation on the idea for chemical chaperone design discussed for lysosomal storage diseases12
, where inhibitors are targeted to improve the delivery of structurally impaired enzymes to the lysosomes. Once delivered to the lysosome, the low pH promotes dissociation of the inhibitor, thereby alleviating any unwanted inhibition while increasing the yield of properly folded enzyme to the lysosomal compartment12
. This work suggests that a rational design for similar molecular chaperones for other mutant enzymes may be possible by exploiting binding of compounds to sites adjacent to the structurally disrupted regions, thus avoiding the possibility of enzymatic inhibition entirely independent of the conditions in which the enzyme operates.