Direct determination of acetyl-enzyme intermediate in the acetylcholinesterase-catalyzed hydrolysis of acetylcholine and acetylthiocholine.

Kinetic characterization of the acyl-enzyme mechanism for β-lactamase I

“…Nor was the use of the indicator to monitor the release of protons on ,6-lactam hydrolysis successful; the rate of reaction at a suitable enzyme concentration (of the order of 100 /LM) was too high for measurement in a stopped-flow spectrophotometer. However, the fact that Froede & Wilson (1984) were able to trap an acyl-enzyme from acetylcholine and acetylcholine esterase prompted our trial of this rather direct method. Moreover, the use of the penamaldate reaction is less costly than the use of relatively large amounts of radioactive substrate.…”

“…The quenched-flow apparatus, modelled after that of Froede & Wilson (1984), comprised three 1 ml Becton Dickinson Plastipak syringes connected via three-way valves. One syringe contained /1-lactamase I (usually about 2,M) and the second contained substrate (e.g.…”

“…Hence k+3, and thus k+2, can be found from measurements of F, when kcat and Km [cf. Froede & Wilson (1984)]. The other parameter found, K', is defined as (k-1 + k+2)/k+,, is referred to in the text as an apparent dissociation constant, and was found as Km (k+2+ k+3)/k+3.…”

Au–Mesoporous silica nanoparticles gated with disulfide-linked oligo(ethylene glycol) chains for tunable cargo delivery mediated by an integrated enzymatic control unit

“…Acetylcholine is also hydrolyzed by the enzyme resulting in the formation of choline and acetic acid. 8 In this case, the release from S3 in the presence of acetylcholine was negligible since the enzymatic process does not yield thiocholine and therefore the MS face remained capped (Figure 4). Release experiments in the presence of sodium iodide showed that the anion iodide (we used acetylthiocholine iodide for the release experiments in Figure 2) had no effect in the response of S3 (Figure 4).…”

“…The Au side (control unit) was functionalized with acetylcholinesterase that is able to detect the presence of acetylthiocholine, a derivative of the neurotransmitter acetylcholine. 7 In particular, the hydrolysis of acetylthiocholine 8 by the enzyme produces thiocholine that cleavages the disulfide bond on the MS face resulting in pore opening and payload release. Moreover, the nanodevice is also sensitive to the presence of diisopropyl fluorophosphate (DFP) which regulates enzyme activity and consequently also cargo delivery from the MS face.…”