Biochemistry 1993, 32, 10414-10422
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Ligand-Dependent Changes in Intrinsic Fluorescence of S-Adenosylhomocysteine Hydrolase: Implications for the Mechanism of Inhibitor-Induced Inhibition? Chong-Sheng Yuan,$ Jerry Yeh,* Thomas C. Squier,* Allen Rawitch,$ and Ronald T. Borchardt*J Department of Biochemistry, The University of Kansas, Lawrence, Kansas 66045, and Department of Biochemistry, The University of Kansas Medical Center, Kansas City, Kansas 66104 Received March 30, 1993; Revised Manuscript Received July 6, 1993”
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-l’-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 fluorescenceand that the enzyme-bound NADH fluorescence was excited by light a t 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. Binding 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 fluorescence in a non-time-dependent manner. However, there was a significant contributiorl to the time-dependent quenching of tryptophan fluorescence that was associated with a conformational change caused by the conversion of the enzyme-bound NAD+ to NADH, as indicated by the observed changes in the solvent accessibility of tryptophan to hydrophilic quenchers (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. Inactivation of the AdoHcy hydrolase (NAD+ form) by mechanism-based inhibitors has been shown previously to involve a cofactor depletion (NAD+ NADH) mechanism. Further evidence in support of this mechanism was obtained when it was observed that, upon inactivation of AdoHcy hydrolase (NAD+ form) with mechanism-based inhibitors, the fluorescence lifetime of tryptophan(s) in AdoHcy hydrolase (NAD+ form) approached the fluorescence lifetime of tryptophan(s) in the N A D H form of the enzyme. When the NADH form of the enzyme was incubated with 3‘-keto-Ado, the fluorescence of the enzyme-bound NADH was almost totally quenched and the intrinsic fluorescence of tryptophan was increased by 15%. A fluorescence method for rapid screening of potential AdoHcy hydrolase inhibitors was developed on the basis of these observations. ABSTRACT:
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S-Adenosylhomocysteine(AdoHcy) hydrolase (EC 3.3.1.1) catalyzes the reversible hydrolysis of AdoHcy to adenosine (Ado) and homocysteine (Hcy) (dela Haba & Cantoni, 1959). This enzyme has been an attractive target for the design of antiviral agents (De Clercq, 1987; Keller & Borchardt, 1988; Wolfe & Borchardt, 1991; Liu et al., 1992a) because of its important role in regulating biological methylation reactions by controlling intracellular levels of AdoHcy (Borchardt, 1980). Inhibition of this enzyme results in intracellular accumulation of AdoHcy and subsequent inhibition of all This work was supported by a grant from the U S . Public Health Service (GM-29332). * To whom correspondenceshould be addressed, at the Department of Pharmaceutical Chemistry, Room 3006, Malott Hall, The University of Kansas, Lawrence, KS 66045. Tel (913) 864-3427; Fax (913) 8425612. *The University of Kansas. f The University of Kansas Medical Center. e Abstract published in Advance ACS Abstracts, September 1 , 1993.
S-adenosylmethionine-dependentmethylation reactions including viral mRNA methylations (Pugh et al., 1978; Borchardt & Pugh, 1979; Keller & Borchardt, 1986; Ransohoff et al., 1987; Hasobe et al., 1989). In recent years, significant efforts have been made in designing potent and selective inhibitorsof AdoHcy hydrolase (Borcherding et al., 1987, 1988; Madhavan et al., 1988; McCarthyet al., 1989; Wolfe & Borchardt, 1991; Jarvi et al., 1991; Robins et al., 1992; Liu et al., 1992a,b; Wolfe et al., 1992; Ramesh et al., 1992; Liu et al., 1993). The design of these potent and specific inhibitors of AdoHcy hydrolase has proceeded in spite of the lack of detailed information about the tertiary structure and the dynamics of this enzyme. Recently, progress has been made toward elucidation of the primary sequence of AdoHcy hydrolase (Ogawa et al., 1987; Coulter-Karis & Hershfield, 1989) and toward identification of key amino acid residues involved in substrate binding and/
0006-2960/93/0432-10414$04.00/0 0 1993 American Chemical Society
Fluorescence of S-Adenosylhomocysteine Hydrolase or catalysis (Aiyar & Hershfield, 1985; Gomi et al., 1986). Yeh et al. (1991) have taken advantage of this information to develop a molecular model of the active site and the NAD+ binding site of the enzyme. This model proposes the existence of several tryptophan residues in the NAD+ binding site of the enzyme. Therefore, we decided to undertake a series of experiments designed to determine whether inhibitor binding and/or enzyme inactivation resulted in changes in the intrinsic fluorescence of these tryptophan residues. Intrinsic protein fluorescence due to endogenous tryptophan(s) is known to be sensitive to local microenvironments and has served as a useful probe for studying protein tertiary structure and its dynamics (Chen & Edelhoch, 1975). Measurement of the changes in intrinsic tryptophan fluorescence in a protein associated with the binding of ligands can be the most direct method to study ligand-induced conformational changes. The kinetic mechanism of AdoHcy hydrolase has been investigated recently using stopped-flow and intrinsic fluorescence quenching techniques (Porter & Boyd, 1991). In the present paper, the ligand-dependent intrinsic fluorescence changes in different forms of AdoHcy hydrolase (apo, NAD+, and NADH) were studied to further demonstrate the mechanism of the enzyme inhibition, as well as to help refine the model for the tertiary structure around both the active site and the NAD+ binding site. MATERIALS AND METHODS
Materials. &Ado, a-Ado, AdoHcy, and Ado deaminase were purchased from Sigma Chemical Co. (St. Louis, MO). The inhibitors, ( l’R,2’S,3’R)-9-(2’,3’-dihydroxycyclopentan1’-y1)adenine (DHCaA), 3-deaza-DHCaA, 4’-a-methyl-DHCaA, 4’-/3-methyl-DHCaA, 4’-fl-vinyl-DHCaA, Ado 5’carboxaldehyde, ( 1’R,2’S,3’R)-9-(2’,3’-dihydroxycyclopent4’-eny1)adenine (DHCeA), and 3-deaza-DHCeA, were synthesized in our laboratory according to published procedures (Borcherding et al., 1987;Narayanan et al., 1988; Wolfe et al., 1992; Liu et al., 1993). (2)-4’,5’-Didehydro-5’-deoxy5’-fluoro-Ado (ZDDFA) was obtained from Dr. James R. McCarthy, Marion Merrell Dow Research Institute, Cincinnati, OH. 2’-Deoxy-2’-methylene-Ado (DOMA), (2)4’,5’-didehydro-5’-deoxy-5’-chloro-Ado(ZDDCA), 5‘-fluoro5’-S-(4-chlorophenyl)-5’-thio-Ado (5‘R/5’S = 61/39) (FSCTAa), 5’-fluoro-5’-S-(4-chlorophenyl)-5’-thio-Ado(5’R/ 5’s= 38/62) (FSCTAb), and 3’-deoxy-3’-amino-Ado and 3’-deoxy-3’-cyclopropyl-Adowere obtained from Dr. Morris Robins, Department of Chemistry, Brigham Young University, Provo, UT. Purification of Recombinant Rat Liver AdoHcy Hydrolase. The recombinant rat liver AdoHcy hydrolase (NAD+ form) was purified from the cell-free extracts of Escherichia coli transformed with plasmid pPUCSAH and grown in the presence of isopropyl P-thiogalactopyranosideby the procedure of Gomi et al. (1989a), except that DEAE-Sepharose instead of DEAE-cellulosewas used for ion-exchangechromatography (Yuan et al., unpublished experiments). The purified enzyme was homogeneous on SDS-PAGE and had a specific activity of 0.9-1.0 unit/mg. The purified enzyme contained 3.7 mol of tightly bound NAD+, 0.3 mol of NADH per tetramer, and six tryptophan residues distributed throughout its 43 1-residue primary sequence (Ogawa et al., 1987). Preparation of Apo-AdoHcy Hydrolase. Apo-AdoHcy hydrolase was obtained by treatment of AdoHcy hydrolase (NAD+ form) with (NH4)2S04 to remove the enzyme-bound NAD+. This method was a modification of that reported for the preparation of rat liver apo-AdoHcy hydrolase (Gomi et
Biochemistry, Vol. 32, No. 39, 1993
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al., 1989b). In this experiment, 12 mg of recombinant rat liver AdoHcy hydrolase (NAD+ form) was dissolved in 4 mL of 25 mM potassium phosphate (K2HP04/KH2P04) buffer, pH 7.2, containing 5 mM dithiothreitol (DTT) and 1 mM EDTA, and 8 mL of saturated (NH4)2S04 solution (pH 3.3), prepared by adjusting the pH of the potassium buffer described above with 3 N HzS04, and was gradually added to the enzyme solution. After stirring for 30 min at Oo C, the precipitated apo-AdoHcy hydrolase was collected by centrifugation and dissolved in 4 mL of potassium phosphate buffer. The apo enzyme was precipitated again by addition of 8 mL of pH 3.3 (NH4)2S04 solution. The precipitate was dissolved in 4 mL of potassium phosphate buffer followed by washing with 8 mL of saturated neutral (NH4)2S04 containing 5 mM DTT and 1 mM EDTA. Finally, the precipitate was dissolved in 25 mM potassium phosphate buffer, pH 7.2, containing 1 mM EDTA. Insoluble matter was removed by centrifugation, and the supernatant was apo-AdoHcy hydrolase. The apo enzyme prepared in the above procedure was inactive in the standard assay, but was converted to active enzyme by preincubation with 1 mM NAD+ for 10 min at room temperature. The specific activity of the enzyme reconstituted with NAD+ was typically 100-120% of that of the recombinant AdoHcy hydrolase (NAD+ form). Presumably, recovery of greater than 100% activity was due to removal of any NADH form of the enzyme generated in the preparation and reconstitution of all the apo-AdoHcy hydrolaseto active enzymewith NAD+. Preparation of AdoHcy Hydrolase (NADH Form). AdoHcy hydrolase (NADH form) was prepared by incubation of the above-prepared apo-AdoHcy hydrolase with 1 mM NADH for 30 min at room temperature. Excess NADH was removed by gel filtration on a Sephadex G-75 column (0.8 X 30cm) equilibrated with 25 mM potassium phosphate buffer, pH 7.2, containing 1 mM EDTA. This NADH form of AdoHcy hydrolase was inactive in the standard assay for AdoHcy hydrolase activity. Assay of Enzyme Activity and Determination of EnzymeBound NAD+ and NADH Content. The activity of AdoHcy hydrolase was assayed in the hydrolytic direction using [3,83H]AdoHcyas substrate with the coupling of Ado deaminase as described previously (Matuszewska & Borchardt, 1987). The enzyme-bound NAD+ and NADH content were determined by the fluorescence method as described previously (Hohman et al., 1985; Matuszewska & Borchardt, 1987). Fluorescence Measurements. Fluorescencemeasurements were performed on a Perkin-Elmer-Hitachi 640-40 fluorescence spectrophotometer controlled by a 3600 data system station. Protein fluorescence was excited at 295 nm to minimize the contribution of protein tyrosine residues to the fluorescence (Eftink & Ghiron, 1976). Changes in protein fluorescence were monitored at the emission maximum of 344 nm. The slit widths on both the excitation and emission monochromators were set at 5 nm. Corrections for innerfilter effects due to the addition of ligand were not necessary since spectrophotometric examination of all the Ado analogs at the highest concentrations employed in this study revealed negligible absorption (
