Two partially unfolded states oftorpedo californicaacetylcholinesterase

Abstract

Chemical modification with sulfhydryl reagents of the single, nonconserved cysteine residue CysZ3' in each subunit of a disulfide-linked dimer of Torpedo californica acetylcholinesterase produces a partially unfolded inactive state. Another partially unfolded state can be obtained by exposure of the enzyme to 1-2 M guanidine hydrochloride. Both these states display several important features of a molten globule, but differ in their spectroscopic (CD, intrinsic fluorescence) and hydrodynamic (Stokes radii) characteristics. With reversal of chemical modification of the former state or removal of denaturant from the latter, both states retain their physicochemical characteristics. Thus, acetylcholinesterase can exist in two molten globule states, both of which are long-lived under physiologic conditions without aggregating, and without either intraconverting or reverting to the native state. Both states undergo spontaneous intramolecular thioldisulfide exchange, implying that they are flexible. As revealed by differential scanning calorimetry, the state produced by chemical modification lacks any heat capacity peak, presumably due to aggregation during scanning, whereas the state produced by guanidine hydrochloride unfolds as a single cooperative unit, thermal transition being completely reversible. Sucrose gradient centrifugation reveals that reduction of the interchain disulfide of the native acetylcholinesterase dimer converts it to monomers, whereas, after such reduction, the two subunits remain completely associated in the partially unfolded state generated by guanidine hydrochloride, and partially associated in that produced by chemical modification. It is suggested that a novel hydrophobic core, generated across the subunit interfaces, is responsible for this noncovalent association. Transition from the unfolded state generated by chemical modification to that produced by guanidine hydrochloride is observed only in the presence of the denaturant, yielding, on extrapolation to zero guanidine hydrochloride, a high free energy barrier (ca. 23.8 kcal/mol) separating these two flexible, partially unfolded states.

Thermal denaturation of wild type and mutant recombinant acetylcholinesterase from amphioxus: effects of the temperature of in vitro expression and of reversible inhibitors

“…Thus, it seems to us that the simplest explanation for the biphasic curves is that there are two different folded, active forms of the enzymes produced in different proportions (f1 and f2) in most cases: a stable conformation ( S ) and an unstable conformation ( U ), which then denature to inactive enzyme ( I ) at different rates ( k 1 and k 2 ). Although denaturation can be described as occurring through an intermediate molten globule ( MG ) state or analogous state (Lumry and Eyring, 1954; Eichler et al, 1994; Kreimer et al, 1995; Kreimer et al, 1996; Shin et al, 1997; Morel et al, 1999), we did not use techniques that would have allowed us to identify such a state; thus, we do not include the MG , or analogous, state in our model (although they could be easily incorporated), which is shown below.…”

Existence of two membrane-bound acetylcholinesterases in the honey bee head

“…These results suggest that AChE m1 and AChE m2 present different conformational states. The Torpedo californica AChE possess two partially unfolded states, stable for many hours under physiological conditions (Kreimer et al, 1996). It is not possible to ascertain whether AChE m1 and AChE m2 are in native or partially unfolded states.…”

Stable nitroxyl radicals as pH, thiol and electron transfer probes

“…Berliner et al [48] used the spin label (1-oxyl-2,2,5,5,-tetramethylpyrroline-3-methyl)-methanethiosulfonate (MTSSL) for reversible chemical modification of Cys25 in papain. We used the biradical ~ for the reversible modification of NADPHcytochrome P450 reductase from rat liver [36,49], human hemoglobin [37], Torpedo californica acetylcholinesterase (TcAChE) [50][51][52], TBADH [39,46] and garlic alliinase (L. Weiner et al, unpubl.). Cys 231, a deeply buried residue in TcAChE, was modified by biradical 9 RS-SR. + HS-~< '-RS-S-(~~) +-R-SH (6) to yield catalyticatly inactive species, even though it is not involved in the active site of the enzyme, which is a serine hydrolase [50][51][52].…”