William D. Mallender

Thioflavin T Is a Fluorescent Probe of the Acetylcholinesterase Peripheral Site That Reveals Conformational Interactions between the Peripheral and Acylation Sites

Three-dimensional structures of acetylcholinesterase (AChE) reveal a narrow and deep active site gorge with two sites of ligand binding, an acylation site at the base of the gorge, and a peripheral site near the gorge entrance. Recent studies have shown that the peripheral site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, but the question of whether the peripheral site makes other contributions to the catalytic process remains open. A possible role for ligand binding to the peripheral site that has long been considered is the initiation of a conformational change that is transmitted allosterically to the acylation site to alter catalysis. However, evidence for conformational interactions between these sites has been difficult to obtain. Here we report that thioflavin T, a fluorophore widely used to detect amyloid structure in proteins, binds selectively to the AChE peripheral site with an equilibrium dissociation constant of 1.0 μm. The fluorescence of the bound thioflavin T is increased more than 1000-fold over that of unbound thioflavin T, the greatest enhancement of fluorescence for the binding of a fluorophore to AChE yet observed. Furthermore, when the acylation site ligands edrophonium or m-(N,N,N-trimethylammonio)trifluoroacetophenone form ternary complexes with AChE and thioflavin T, the fluorescence is quenched by factors of 2.7–4.2. The observation of this partial quenching of thioflavin T fluorescence is a major advance in the study of AChE for two reasons. First, it allows thioflavin T to be used as a reporter for ligand reactions at the acylation site. Second, it indicates that ligand binding to the acylation site initiates a change in the local AChE conformation at the peripheral site that quenches the fluorescence of bound thioflavin T. The data provide strong evidence in support of a conformational interaction between the two AChE sites.

Mechanistic Studies of Substrate-assisted Inhibition of Ubiquitin-activating Enzyme by Adenosine Sulfamate Analogues

Ubiquitin-activating enzyme (UAE or E1) activates ubiquitin via an adenylate intermediate and catalyzes its transfer to a ubiquitin-conjugating enzyme (E2). MLN4924 is an adenosine sulfamate analogue that was identified as a selective, mechanism-based inhibitor of NEDD8-activating enzyme (NAE), another E1 enzyme, by forming a NEDD8-MLN4924 adduct that tightly binds at the active site of NAE, a novel mechanism termed substrate-assisted inhibition (Brownell, J. E., Sintchak, M. D., Gavin, J. M., Liao, H., Bruzzese, F. J., Bump, N. J., Soucy, T. A., Milhollen, M. A., Yang, X., Burkhardt, A. L., Ma, J., Loke, H. K., Lingaraj, T., Wu, D., Hamman, K. B., Spelman, J. J., Cullis, C. A., Langston, S. P., Vyskocil, S., Sells, T. B., Mallender, W. D., Visiers, I., Li, P., Claiborne, C. F., Rolfe, M., Bolen, J. B., and Dick, L. R. (2010) Mol. Cell 37, 102–111). In the present study, substrate-assisted inhibition of human UAE (Ube1) by another adenosine sulfamate analogue, 5′-O-sulfamoyl-N6-[(1S)-2,3-dihydro-1H-inden-1-yl]-adenosine (Compound I), a nonselective E1 inhibitor, was characterized. Compound I inhibited UAE-dependent ATP-PPi exchange activity, caused loss of UAE thioester, and inhibited E1-E2 transthiolation in a dose-dependent manner. Mechanistic studies on Compound I and its purified ubiquitin adduct demonstrate that the proposed substrate-assisted inhibition via covalent adduct formation is entirely consistent with the three-step ubiquitin activation process and that the adduct is formed via nucleophilic attack of UAE thioester by the sulfamate group of Compound I after completion of step 2. Kinetic and affinity analysis of Compound I, MLN4924, and their purified ubiquitin adducts suggest that both the rate of adduct formation and the affinity between the adduct and E1 contribute to the overall potency. Because all E1s are thought to use a similar mechanism to activate their cognate ubiquitin-like proteins, the substrate-assisted inhibition by adenosine sulfamate analogues represents a promising strategy to develop potent and selective E1 inhibitors that can modulate diverse biological pathways.

A steric blockade model for inhibition of acetylcholinesterase by peripheral site ligands and substrate.

The active site gorge of acetylcholinesterase (AChE) contains two sites of ligand binding, an acylation site near the base of the gorge and a peripheral site at its mouth. We recently introduced a steric blockade model which demonstrated that small peripheral site ligands like propidium can inhibit substrate hydrolysis simply by decreasing the substrate association and dissociation rate constants without altering the equilibrium constant for substrate binding to the acylation site. We now employ our nonequilibrium kinetic analysis to extend this model to include blockade of the dissociation of substrate hydrolysis products by bound peripheral site ligand. We also report here that acetylthiocholine can bind to the AChE peripheral site with an equilibrium dissociation constant K(S) of about 1 mM. This value was determined from the effect of the acetylthiocholine concentration on the rate at which fasciculin associates with the peripheral site. When substrate binding to the peripheral site is incorporated into our steric blockade model, hydrolysis rates at low substrate concentration appear to be accelerated while substrate inhibition of hydrolysis occurs at high substrate concentration. The model predicts that hydrolysis rates for substrates which equilibrate with the acylation site prior to the acylation step should not be inhibited by bound peripheral site ligand. Organophosphates equilibrate with AChE prior to phosphorylating the active site serine residue, and as predicted propidium had little effect on the phosphorylation rate constants for the fluorogenic organophosphate ethylmethyl-phosphonylcoumarin (EMPC). The 2nd-order phosphorylation rate constant kOP/K(OP) was decreased 3-fold by a high concentration of propidium and the 1st-order rate constant kOP increased somewhat. In contrast to propidium, when the neurotoxin fasciculin bound to the AChE peripheral site both a steric blockade and a conformational change in the acylation site appeared to occur. With saturating fasciculin, kOP/K(OP) decreased by a factor of more than 750 and kOP decreased 300-fold. These data suggest that new peripheral site ligands may be designed to have selective effects on AChE phosphorylation.

Organophosphorylation of Acetylcholinesterase in the Presence of Peripheral Site Ligands DISTINCT EFFECTS OF PROPIDIUM AND FASCICULIN

Structural analysis of acetylcholinesterase (AChE) has revealed two sites of ligand interaction in the active site gorge: an acylation site at the base of the gorge and a peripheral site at its mouth. A goal of our studies is to understand how ligand binding to the peripheral site alters the reactivity of substrates and organophosphates at the acylation site. Kinetic rate constants were determined for the phosphorylation of AChE by two fluorogenic organophosphates, 7-[(diethoxyphosphoryl)oxy]-1-methylquinolinium iodide (DEPQ) and 7-[(methylethoxyphosphonyl)oxy]-4-methylcoumarin (EMPC), by monitoring release of the fluorescent leaving group. Rate constants obtained with human erythrocyte AChE were in good agreement with those obtained for recombinant human AChE produced from a high level Drosophila S2 cell expression system. First-order rate constants k OP were 1,600 ± 300 min−1 for DEPQ and 150 ± 11 min−1 for EMPC, and second-order rate constants k OP/K OP were 193 ± 13 μm −1 min−1 for DEPQ and 0.7–1.0 ± 0.1 μm −1 min−1for EMPC. Binding of the small ligand propidium to the AChE peripheral site decreased k OP/K OPby factors of 2–20 for these organophosphates. Such modest inhibitory effects are consistent with our recently proposed steric blockade model (Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998)Biochemistry 37, 4206–4216). Moreover, the binding of propidium resulted in a clear increase in k OPfor EMPC, suggesting that molecular or electronic strain caused by the proximity of propidium to EMPC in the ternary complex may promote phosphorylation. In contrast, the binding of the polypeptide neurotoxin fasciculin to the peripheral site of AChE dramatically decreased phosphorylation rate constants. Values ofk OP/K OP were decreased by factors of 103 to 105, andk OP was decreased by factors of 300–4,000. Such pronounced inhibition suggested a conformational change in the acylation site induced by fasciculin binding. As a note of caution to other investigators, measurements of phosphorylation of the fasciculin-AChE complex by AChE inactivation gave misleading rate constants because a small fraction of the AChE was resistant to inhibition by fasciculin.

Mechanistic Studies on Activation of Ubiquitin and Di-ubiquitin-like Protein, FAT10, by Ubiquitin-like Modifier Activating Enzyme 6, Uba6

Background: The Uba6 pathway and its components play an important role in a variety of biological processes. Results: The mechanism of how Uba6 activates two distinct substrates, ubiquitin and FAT10, was characterized. Conclusion: Uba6 was shown to use a similar mechanism for activating both substrates with a greater affinity for FAT10. Significance: Relative levels of ubiquitin and FAT10 could regulate the Uba6 pathway in cells. Uba6 is a homolog of the ubiquitin-activating enzyme, Uba1, and activates two ubiquitin-like proteins (UBLs), ubiquitin and FAT10. In this study, biochemical and biophysical experiments were performed to understand the mechanisms of how Uba6 recognizes two distinct UBLs and catalyzes their activation and transfer. Uba6 is shown to undergo a three-step activation process and form a ternary complex with both UBLs, similar to what has been observed for Uba1. The catalytic mechanism of Uba6 is further supported by inhibition studies using a mechanism-based E1 inhibitor, Compound 1, which forms covalent adducts with both ubiquitin and FAT10. In addition, pre-steady state kinetic analysis revealed that the rates of UBL-adenylate (step 1) and thioester (step 2) formation are similar between ubiquitin and FAT10. However, distinct kinetic behaviors were also observed for ubiquitin and FAT10. FAT10 binds Uba6 with much higher affinity than ubiquitin while demonstrating lower catalytic activity in both ATP-PPi exchange and E1-E2 transthiolation assays. Also, Compound 1 is less potent with FAT10 as the UBL compared with ubiquitin in ATP-PPi exchange assays, and both a slow rate of covalent adduct formation and weak adduct binding to Uba6 contribute to the diminished potency observed for FAT10. Together with expression level analysis in IM-9 cells, this study sheds light on the potential role of cytokine-induced FAT10 expression in regulating Uba6 pathways.

Substrate Binding to the Peripheral Site Occurs on the Catalytic Pathway of Acetylcholinesterase and Leads to Substrate Inhibition

Two sites of ligand interaction in acetylcholinesterase (AChE) were first demonstrated in ligand binding studies (7) and later confirmed by crystallography, site-specific mutagenesis and molecular modeling: an acylation site at the base of the gorge, and a peripheral site at its mouth. Here we address the question of how ligand binding to the peripheral site alters AChE catalytic activity. In the traditional AChE catalytic pathway (Scheme 1 below), the initial enzyme-substrate complex ES proceeds to an acylated enzyme intermediate EA which is then hydrolyzed to product P and E (2). When a peripheral site inhibitor (I) is included, typical analysis of inhibition patterns assumes S and I binding are at equilibrium (3; 4). We avoid these equilibrium assumptions here for the first time in the AChE literature by solving the appropriate differential rate equations with the simulation program SCoP. In particular, we examine the simple hypothesis that the only effect of peripheral site inhibitors like propidium is to impose a steric blockade that decreases association and dissociation rate constants for substrates and other acylation site ligands without altering their ratio, the equilibrium association constant. We test this hypothesis by examining the effect of peripheral site ligands on the rate constants for the binding of acylation site inhibitors and on the inhibition of substrate hydrolysis. We turn next to the questions of whether acetylthiocholine itself can bind to the peripheral site and whether this binding is of significance on the catalytic pathway. The acetylthiocholine affinity for the peripheral site is determined by measuring acetylthiocholine inhibition of the association rate k on for the slowly equilibrating peripheral site ligand fasciculin 2. Our re-suits indicate that failure to achieve equilibrium has a profound impact on the classical interpretation of AChE inhibition and indeed alters mechanistic conclusions. A physiological role of substrate binding to the peripheral site in the catalytic pathway is also suggested.

Substrate Binding to the Acetylcholinesterase Peripheral Site Promotes Substrate Hydrolysis but also Gives Rise to Substrate Inhibition

Many ligands that bind to the peripheral site of acetylcholinesterase (AChE) inhibit substrate hydrolysis at the acylation site, though the interpretation of this inhibition has been unclear. Since previous explanations based upon equilibrium ligand binding cannot be justified for AChE, we introduced an alternative nonequilibrium analysis. This analysis incorporates a steric blockade mechanism which assumes that the only effects of a small bound peripheral ligand are to slow down the rates of substrate association and dissociation at the acylation site and concomitantly reduce the product dissociation rate. Experimental data using propidium and gallamine as peripheral site ligands and acetylthiocholine, phenyl acetate, huperzine A and m-((N, N, N -trimethylammonio)-trifluoroacetophenone as acylation site ligands agreed well with our theory. The nonequilibrium model has been extended to account for substrate inhibition based upon acetylthiocholine affinity for the peripheral site. This affinity was determined by measuring acetylthiocholine inhibition of the association rate k on for the slowly equilibrating peripheral site ligand fasciculin 2. The measured K s of about 1–2 mM was considerably lower than the K ss estimate of about 20 mM determined by application of the Haldane equation to the steady-state substrate inhibition curve. A better understanding of the interaction between peripheral site ligands and acetylcholinesterase may help to understand the physiological role of the peripheral site and to design new ligands that inhibit organophosphorylation.

Cyclic, Selectively Permeable Acetylcholinesterase Inhibitors

An area of AChE research of great interest is the development of new drugs for the prevention and treatment of enzyme inactivation by organophosphates (compounds found in both pesticides and chemical warfare agents). Currently, there are compounds available to reactivate organophosphate-neutralized AChE, but no compounds exist for pre-treatment to actively protect AChE during exposure to organophosphate agents. In our studies, molecular modeling has been used to examine the interactions between AChE and various currently known inhibitors to design new peripheral site inhibitors. Such modeling efforts are possible due to the availability of three-dimensional crystal structures of unliganded AChE and the fasciculin-AChE complex. Fasciculin (Fas), a snake venom neurotoxic peptide, binds to the peripheral site of AChE and inhibits enzyme activity by a mechanism involving steric blockade and conformational change in the enzyme active site. With the information from these models, novel cyclic peptide and pseudopeptide compounds are being designed and assayed for selective binding to the peripheral site of AChE. The initial screening protocol includes assays for inhibition of substrate hydrolysis by AChE and for competition with Fas for binding to the AChE peripheral site. The goal of this project is to identify new peripheral site ligands that selectively inhibit AChE phosphorylation while permitting sufficient acetylcholine hydrolysis to maintain physiologic function. Both combinatorial libraries and rationally designed compounds are being screened in the hopes of identifying candidate molecules for future refinement through molecular docking and modeling simulations into lead drug compounds.