Geometric isomers of a photoactivable general anesthetic delineate a binding site on adenylate kinase.
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 approximately 5 A 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%.
Addona
George H
GH
Department of Anesthesia and Critical Care, Massachusetts General Hospital, Boston 02114, USA.
Husain
S Shaukat
SS
Stehle
Thilo
T
Miller
Keith W
KW
D000263:Adenylate Kinase / Q000378:metabolism*; D018681:Anesthetics, General / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D002853:Chromatography, Liquid; D007536:Isomerism; D013058:Mass Spectrometry; D008958:Models, Molecular; D010777:Photochemistry
D018681:Anesthetics, General; D000263:Adenylate Kinase