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)