Time-resolved fluorescence spectroscopy of human adenosine deaminase: effects of enzyme inhibitors on protein conformation.
Adenosine deaminase, a purine salvage enzyme essential for immune competence, was studied by time-resolved fluorescence spectroscopy. The heterogeneous emission from this four-tryptophan protein was separated into three lifetime components: tau 1 = 1 ns and tau 2 = 2.2 ns an emission maximum at about 330 nm and tau 3 = 6.3 ns with emission maximum at about 340 nm. Solvent accessibility of the tryptophan emission was probed with polar and nonpolar fluorescence quenchers. Acrylamide, iodide, and trichloroethanol quenched emission from all three components. Acrylamide quenching caused a blue shift in the decay-associated spectrum of component 3. The ground-state analogue enzyme inhibitor purine riboside quenched emission associated with component 2 whereas the transition-state analogue inhibitor deoxycoformycin quenched emission from both components 2 and 3. The quenching due to inhibitor binding had no effect on the lifetimes or emission maxima of the decay-associated spectra. These observations can be explained by a simple model of four tryptophan environments. Quenching studies of the enzyme-inhibitor complexes indicate that adenosine deaminase undergoes different protein conformation changes upon binding of ground- and transition-state analogue inhibitors. The results are consistent with localized structural alterations in the enzyme.
Philips
A V
AV
Department of Biochemistry, University of Kentucky, Lexington 40536-0084.
Coleman
M S
MS
Maskos
K
K
Barkley
M D
MD
D020106:Acrylamide; D000178:Acrylamides / Q000494:pharmacology; D000243:Adenosine Deaminase; D058892:Adenosine Deaminase Inhibitors; D055598:Chemical Phenomena; D002621:Chemistry; D015200:Fluoroimmunoassay / Q000379:methods; D006801:Humans; D009700:Nucleoside Deaminases / Q000037:antagonists & inhibitors; D011487:Protein Conformation / Q000187:drug effects; D013052:Spectrometry, X-Ray Emission; D013997:Time Factors
D000178:Acrylamides; D058892:Adenosine Deaminase Inhibitors; D020106:Acrylamide; D009700:Nucleoside Deaminases; D000243:Adenosine Deaminase