Ligand-dependent changes in intrinsic fluorescence of S-adenosylhomocysteine hydrolase: implications for the mechanism of inhibitor-induced inhibition.
Different forms of S-adenosylhomocysteine (AdoHcy) hydrolase (NAD+, apo, and NADH forms) were prepared, and the effects of ligand binding on the intrinsic tryptophan fluorescence were investigated. Binding of AdoHcy hydrolase (NAD+ form) with its mechanism-based inhibitors [e.g., (1'R,2'S,3'R)-9-(2',3'-dihydroxycyclopentan-1'-yl)adenine] caused significant quenching (35%) of the intrinsic tryptophan fluorescence of the enzyme. A ligand-induced conformational change in the tertiary structure of the enzyme accounted for an initial 10% quenching of the fluorescence, with further fluorescence quenching occurring in a time-dependent manner. This time-dependent quenching of fluorescence was consistent with the time-dependent inactivation of the enzyme and the time-dependent reduction of the enzyme-bound NAD+ to NADH. The time-dependent quenching of the intrinsic tryptophan fluorescence is largely the result of resonance energy transfer from tryptophan(s) in the enzyme to the enzyme-bound NADH. This interpretation is supported by the observations that the formation of enzyme-bound NADH quenched the intensity of the intrinsic tryptophan fluorescence and that the enzyme-bound NADH fluorescence was excited by light at wavelengths consistent with the absorption spectrum of tryptophan. Additional support for the involvement of NADH in the time-dependent tryptophan fluorescence quenching came from the observation that this quenching could only be observed when binding caused simultaneous reduction of the enzyme-bound NAD+ to NADH.(ABSTRACT TRUNCATED AT 250 WORDS)
Yuan
C S
CS
Department of Biochemistry, University of Kansas, Lawrence 66045.
Yeh
J
J
Squier
T C
TC
Rawitch
A
A
Borchardt
R T
RT
D043383:Adenosylhomocysteinase; D000818:Animals; D001665:Binding Sites; D003198:Computer Simulation; D004735:Energy Transfer; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000201:enzymology; D008958:Models, Molecular; D009243:NAD / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D008024:Ligands; D009243:NAD; D014364:Tryptophan; D006867:Hydrolases; D043383:Adenosylhomocysteinase