Geometric Isomers of a Photoactivable General Anesthetic Delineate a Binding Site on Adenylate Kinase

Abstract

General anesthetics are a class of drugs whose mode of action is poorly understood. Here, two photoactivable general anesthetics, n-octan-1-ol geometric isomers bearing a diazirine group on either the third or seventh carbon (3-and 7-azioctanol, respectively), were used to locate and delineate an anesthetic site on adenylate kinase. Each photoincorporated at a mole ratio of 1:1 as determined by mass spectrometry. The photolabeled kinase was subjected to tryptic digest, and the fragments were separated by chromatography and sequenced by mass spectrometry. 3-Azioctanol photolabeled His-36, whereas its isomer, 7-azioctanol, photolabeled Asp-41. Inspection of the known structure of adenylate kinase shows that the side chains of these residues are within ϳ5 Å of each other. This distance matches the separation of the 3-and 7-positions of an extended aliphatic chain. The alkanol site so-defined spans two domains of adenylate kinase. His-36 is part of the CORE domain, and Asp-41 belongs to the nucleotide monophosphate binding domain. Upon ligand binding the nucleotide monophosphate binding domain rotates relative to the CORE domain, causing a conformational change that might be expected to affect alkanol binding. Indeed, the substrate-mimicking inhibitor adenosine-(5)-pentaphospho-(5)-adenosine (Ap5A) reduced the photoincorporation of 3-[ 3 H]azioctanol by 75%.

Identification of Binding Sites in the Nicotinic Acetylcholine Receptor for [3H]Azietomidate, a Photoactivatable General Anesthetic

“…However, the selective labeling of a subset of Tyr, Glu, or Asp within the ACh binding sites or at the entry to the ion channel indicate that the labeling results from occupancy of binding sites and not diffusional encounters with generally reactive side chains. Further evidence consistent with this conclusion is provided by studies with adenylate kinase, where 3-azioctanol reacted with a His and 7-azioctanol reacted with an Asp that in the crystal structure are in a pocket within 5 Å of each other (42).…”

Identification of a General Anesthetic Binding Site in the Diacylglycerol-binding Domain of Protein Kinase Cδ

“…It is not possible from mass spectrometry alone to establish the percentage of PKC␦ C1B that was photolabeled, but, based on previous experience with adenylate kinase, it is unlikely to be high (26). Consequently, as discussed previously (26), it is difficult to rule out the existence of fractions photoincorporating two alcohols per PKC␦ C1B because of the predicted low probability of such species occurring. However, two observations make this unlikely.…”

“…No higher molecular weight peak corresponding to the photoincorporation of two azioctanols (7,619 Da) was observed. Nonetheless, it is hard to rule this out because the probability of such labeling is likely to be low (26). A major unidentified contaminant occurred at 7458 Da, but only in this particular preparation.…”

An alcohol binding site on the neural cell adhesion molecule L1

“…Azialcohols, bearing a photolabile diazirine moiety on one carbon, are stable general anesthetics with normal potencies that obey the MeyerOverton rule. Since their introduction (23), azialcohols have been used to locate binding sites on the nicotinic acetylcholine receptor (24), protein kinase C (25), and adenylate kinase (26).…”