Interaction of Adenosine Deaminase with Inhibitors. Chemical Modification by Diethyl Pyrocarbonate

S. S. Mardanyan^1*, S. G. Sharoyan¹, A. A. Antonyan¹, G. Lupidi², and G. Cristalli³

¹Buniatyan Institute of Biochemistry, Armenian National Academy of Sciences, ul. P. Sevaka 5/1, Yerevan, 375014 Republic of Armenia; fax: (374-1) 281-634; E-mail: biochem@ipia.sci.am

²Department of MCA Sciences, University of Camerino, Via Camerini 2, 62032 Camerino (MC), Italy; E-mail: giulio.lupidi@unicam.it

³Department of Chemical Sciences, University of Camerino, Via S. Agostino 1, 62032 Camerino (MC), Italy; fax: (39-0737) 402-252; E-mail: gloria.cristalli@unicam.it

^* To whom correspondence should be addressed.

Received June 8, 2001; Revision received March 26, 2002

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The interaction of adenosine deaminase (adenosine aminohydrolase, ADA) from bovine spleen with inhibitors--erythro-9-(2-hydroxy-3-nonyl)adenine, erythro-9-(2-hydroxy-3-nonyl)-3-deazaadenine, and 1-deazaadenosine--was investigated. Using selective chemical modification by diethyl pyrocarbonate (DEP), the possible involvement of His residues in this interaction was studied. The graphical method of Tsou indicates that of six His residues modified in the presence of DEP, only one is essential for ADA activity. Inactivation of the enzyme, though with low rate, in complex with any of the inhibitors suggests that the adenine moiety of the inhibitors (and consequently, of the substrate) does not bind with the essential His to prevent its modification. The absence of noticeable changes in the dissociation constants of any of the enzyme-inhibitor complexes for the DEP-modified and control enzyme indicates that at least the most available His residues modified in our experiments do not participate in binding the inhibitors--derivatives of adenosine or erythro-9-(2-hydroxy-3-nonyl)adenine.

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KEY WORDS: adenosine deaminase, active site, histidine residues, chemical modification, adenosine deaminase inhibitors

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