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+, ap o, and NADH forms) were prepared, and the effects of ligand binding on the intrinsic tryptophan fluorescence were investigated. Binding of A doHcy hydrolase (NAD+ form) with its mechanism-based inhibitors [e.g., ,3'R)-9-(2',3'-dihydroxycyclopentan-1'-yl)adenine] caused significant quenching (35%) of the intrinsic tryptophan fluorescence of the enzym e. 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 ma nner. This time-dependent quenching of fluorescence was consistent wit h the time-dependent inactivation of the enzyme and the time-dependent reduction of the enzyme-bound NAD+ to NADH. The time-dependent quench ing of the intrinsic tryptophan fluorescence is largely the result of resonance energy transfer from tryptophan(s) in the enzyme to the enzy me-bound NADH. This interpretation is supported by the observations th at the formation of enzyme-bound NADH quenched the intensity of the in trinsic tryptophan fluorescence and that the enzyme-bound NADH fluores cence was excited by light at wavelengths consistent with the absorpti on spectrum of tryptophan. Additional support for the involvement of N ADH in the time-dependent tryptophan fluorescence quenching came from the observation that this quenching could only be observed when bindin g caused simultaneous reduction of the enzyme-bound NAD+ to NADH. Bind ing of apo-AdoHcy hydrolase with mechanism-based inhibitors or binding of AdoHcy hydrolase (NAD+ form) with 3'-keto-Ado or other 3'-modified Ado analogs that were incapable of reducing the enzyme-bound NAD+ to NADH resulted in only 10% quenching of the intrinsic tryptophan fluore scence in a non-time-dependent manner. However, there was a significan t, contribution to the time-dependent, quenching of tryptophan fluores cence that was associated with a conformational change caused by the c onversion of the enzyme-bound NAD+ to NADH, as indicated by the observ ed changes in the solvent accessibility of tryptophan to hydrophilic q uenchers (e.g., KI) that varied between the NAD+ and the apo forms of the enzyme after binding with the mechanism-based inhibitor. Data also indicated that there was at least one tryptophan near the active site of the enzyme and two tryptophans near the NAD+ binding site. Inactiv ation of the AdoHcy hydrolase (NAD+ form) by mechanism-based inhibitor s has been shown previously to involve a cofactor depletion (NAD+ --> NADH) mechanism. Further evidence in support of this mechanism was obt ained when it was observed that, upon inactivation of AdoHcy hydrolase (NAD+ form) with mechanism-based inhibitors, the fluorescence lifetim e of tryptophan(s) in AdoHcy hydrolase (NAD+ form) approached the fluo rescence lifetime of tryptophan(s) in the NADH form of the enzyme. Whe n the NADH form of the enzyme was incubated with 3'-keto-Ado, the fluo rescence of the enzyme-bound NADH was almost totally quenched and the intrinsic fluorescence of tryptophan was increased by 15%. A fluoresce nce method for rapid screening of potential AdoHcy hydrolase inhibitor s was developed on the basis of these observations.