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Biochemistry 1996, 35, 2658-2667
Affinity Labeling of the Active Site of Rabbit Muscle Adenylosuccinate Lyase by 2-[(4-Bromo-2,3-Dioxobutyl)Thio]Adenosine 5′-Monophosphate† Sadanand U. Gite and Roberta F. Colman* Department of Chemistry and Biochemistry, UniVersity of Delaware, Newark, Delaware 19716 ReceiVed NoVember 6, 1995; ReVised Manuscript ReceiVed December 21, 1995X
ABSTRACT: Rabbit muscle adenylosuccinate lyase upon incubation with 7.5-50 µM 2-[(4-bromo-2,3dioxobutyl)thio]adenosine 5′-monophosphate (2-BDB-TAMP) in 0.05 M PIPES buffer, pH 7.0 and 10 °C, gives a time dependent biphasic inactivation. The rate of inactivation exhibits a nonlinear dependence on the concentration of 2-BDB-TAMP, which can be described by reversible binding of reagent to the enzyme (KI ) 8.5 µM, 5.2 µM) prior to the irreversible reaction, with maximum rate constants of 0.319 and 0.027 min-1 for the fast and slow phases, respectively. The enzyme is a tetramer, with subunits of 50 000 Da. When the enzyme was 90% inactivated, 0.84 mol of reagent/mol of subunit was incorporated as measured by protein-bound phosphate analysis; similar results were obtained using 2-BDB-[14C]TAMP. Complete protection against inactivation and incorporation was afforded by 1 mM 5′-AMP and by 0.1 mM 5′-AMP + 5 mM fumarate (the natural products of adenylosuccinate hydrolysis) but not by 0.1 mM 5′-AMP alone, 5 mM fumarate alone, or 0.1 mM 5′-AMP + 5 mM maleate or 5 mM succinate. These studies suggest that 2-BDB-TAMP inactivates adenylosuccinate lyase by specific reaction at the substrate binding site, with negative cooperativity between subunits accounting for the appearance of two phases of inactivation. Cleavage of 2-BDB-TAMP-modified enzyme with cyanogen bromide and subsequent separation of peptides by reverse phase HPLC gave only one radioactive peak. This radioactive peptide was further digested with papain and the target site of the 2-BDB-TAMP reaction was identified as Arg112. We conclude that Arg112 is located in the substrate binding site of rabbit muscle adenylosuccinate lyase.
Adenylosuccinate lyase [EC 4.3.2.2] catalyzes the conversion of adenylosuccinate to 5′-AMP and fumarate, a critical reaction in the purine biosynthesis pathway (Miller et al., 1957; Ratner, 1973). Deficiency of adenylosuccinate lyase activity is associated with severe mental retardation and secondary autistic features in a number of human patients (Jaeken & Van den Berghe, 1984; Van den Berghe & Jaeken et al., 1986; Jaeken et al., 1988, 1992; Barshop et al., 1989; Van den Berghe et al., 1991). In one family, the deficiency was traced to a single mutation (Ser413Pro) which results in structural instability of the enzyme rather than a change in catalysis (Stone et al., 1992). Kinetic studies have been conducted on the enzyme from a variety of sources, including yeast (Cohen & Bridger, 1964; Bridger & Cohen, 1968, 1969), rat (Casey & Lowenstein, 1987), chicken (Aimi et al., 1990), and human (Stone et al., 1993). Adenylosuccinate lyases from several species are homotetramers with subunits of about 50 000 Da (Casey & Lowenstein, 1987; Stone et al., 1993). The cDNAs of adenylosuccinate lyase from Bacillus subtilis (Ebbole & Zalkin, 1987), chicken (Aimi et al., 1990), and human (Stone et al., 1993) have been cloned, and their amino acid sequences have been derived from their corresponding cDNA sequences. All three exhibit considerable resemblance, and the amino acid sequences of the enzymes from chicken and human show a high degree of identity (85%) plus similarity (5%). Although Stone et al. (1993) have postulated a mechanism involving general acid and base catalysis in the cleavage of adenylosuccinate, no enzymatic amino acids have †
This work was supported by NSF Grant MCB-94-23108. * To whom correspondence should be addressed. X Abstract published in AdVance ACS Abstracts, February 1, 1996.
0006-2960/96/0435-2658$12.00/0
been directly implicated in either catalysis or substrate binding. Affinity labeling provides a powerful approach to identify amino acid residues within specific binding sites in enzymes (Colman, 1983, 1989, 1990, 1995). In the present study, 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5′-monophosphate (2-BDB-TAMP)1 has been selected as a potential affinity label for adenylosuccinate lyase because of its structural similarity to the substrate, adenylosuccinate (Figure 1). Reaction of 2-BDB-TAMP can potentially occur with the side chains of several amino acids of proteins, including Lys, Arg, Cys, His, Glu, Asp, Tyr, and Met (Colman, 1989, 1990, 1995; Kapetanovic et al., 1985). Such reactions can occur at the carbonyl groups or by nucleophilic displacement of the bromide from -CH2Br. Thus, once the nucleotide derivative is bound at a specific site, it is likely to have an accessible amino acid target. In addition, 2-BDB-TAMP is soluble in water, eliminating the need for addition of organic solvent in the reaction mixture. Previously, in this laboratory, bromodioxobutyl nucleotides have been shown to function as affinity labels of several enzymes including pyruvate kinase (Kapetanovic et al., 1985; DeCamp et al., 1988), isocitrate dehydrogenase (Huang & Colman, 1984) and glutamate dehydrogenase (Batra & Colman, 1984, 1986). 1 Abbreviations: 2-BDB-TAMP, 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5′-monophosphate; DTT, dithiothreitol; HPLC, highperformance liquid chromatography; NEM, N-ethylmaleimide; TFA, trifluoroacetic acid; PIPES, piperazine-N,N′-bis[2-ethanesulfonic acid]; HEPES, N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]; FPLC, fast protein liquid chromatography; MES, 2-[N-morpholino]ethanesulfonic acid.
© 1996 American Chemical Society
Affinity Labeling of Adenylosuccinate Lyase
FIGURE 1: Schematic comparison of the structures of the substrate, adenylosuccinate, and 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5′-monophosphate.
We describe here the specific modification of rabbit muscle adenylosuccinate lyase by the nucleotide affinity label 2-BDB-TAMP. A preliminary version of this work has been presented (Gite & Colman, 1995). EXPERIMENTAL PROCEDURES Materials. Rabbit muscle (mature) was purchased from Pel-Freez Biologicals and stored frozen at -80 °C until further use. Adenylosuccinate, fumarate, succinate, maleate, 5′-AMP, dithiothreitol (DTT), N-ethylmaleimide (NEM), Sephadex G-10, papain, and phosphate standard solution were all obtained from Sigma Chemical Co. Carbon disulfide, chloroperoxybenzoic acid, and 1,4-dibromobutanedione were from Aldrich Chemical Co. Prior to use, 1,4-dibromobutanedione was recrystallized from petroleum ether. DEAE-cellulose (DE-52) was supplied by Whatman, Matrex Blue by Amicon, and molecular weight marker kits by Pharmacia Fine Chemicals. Cyanogen bromide was obtained from Fluka, and ammonium sulfate (ultrapure) and protease from Staphylococcus aureus, strain V8, were purchased from ICN Biochemicals. The Bio-Rad protein assay dye reagent was supplied by Bio-Rad. 5′-[14C]AMP (generally labeled) and [3H]NaBH4 were obtained from New England Nuclear-DuPont. All other chemicals used were the highest purity reagent grade. The commercially available papain, obtained from Sigma Chemical Co., exhibited numerous peptide peaks when subjected to HPLC on a C18 column. Thus, the papain preparation requires additional purification prior to use. Papain (20 mg) was purified by HPLC on a Varian model 5000 HPLC system equipped with a reverse phase Vydac C18 column (0.46 × 25 cm). The proteolytic enzyme was dissolved in 1 mL of 0.02 M ammonium acetate buffer (pH 6.0), centrifuged at 14 000 rpm for 5 min, and injected onto the column equilibrated with solvent A (0.02 M ammonium acetate, pH 6.0). The column was eluted with solvent A for 10 min, followed by a linear gradient from solvent A to 100% solvent B (0.02 M ammonium acetate, pH 6.0, in 50% acetonitrile) between 10 and 110 min. The flow rate was 1 mL/min. The effluent was continuously monitored at 280 nm, and fractions of 1 mL were collected. Fractions 54-
Biochemistry, Vol. 35, No. 8, 1996 2659 57 were pooled and lyophilized; this purified papain preparation was used to digest adenylosuccinate lyase fragments. Preparation of 2-[(4-Bromo-2,3-dioxobutyl)thio]adenosine 5′-Monophosphate. 2-BDB-TAMP was synthesized from 5′AMP according to the procedure of Kapetanovic et al. (1985). The concentration of the final compound was calculated from its UV spectrum in 0.1 M MES buffer, pH 6.0, using 246 nm ) 35 200 and 270 nm ) 11 500 M-1 cm-1. The synthesis of 2-BDB-[14C]TAMP was conducted by the same method using as starting material 110 µCi of generally labeled 5′-AMP plus 0.74 mmol of non-radioactive 5′-AMP. The overall yield of 2-BDB-TAMP was 10%-20%. The specific radioactivity of 2-BDB-[14C]TAMP was about 3.2 × 1011 cpm/mol. Enzyme Assay and Protein Determination. The adenylosuccinate lyase activity of the enzyme was measured spectrophotometrically using a double-beam Perkin-Elmer spectrophotometer equipped with a recorder set to 0.1 absorbance full scale. The assay was performed at 25 °C in a total volume of 1 mL containing 30 µM adenylosuccinate in 10 mM Tris-HCl buffer (pH 7.4), 0.15 M NaCl, and appropriately diluted enzyme (1-5 µg). The hydrolysis of adenylosuccinate was followed by the decrease in absorbance at 282 nm using the difference extinction coefficient of 10 000 M-1 cm-1 between the substrate, adenylosuccinate, and the product, 5′-AMP (Tornheim & Lowenstein, 1972). One unit of enzyme activity is defined as the amount of enzyme that converts one micromole of adenylosuccinate to 5′-AMP and fumarate per minute at 25 °C, and the specific activity of the enzyme is taken as the enzyme units per milligram of protein. For the purified enzyme, the protein concentration was determined according to the method of Groves et al. (1968) from the difference between A224 nm and A233 nm using crystalline bovine serum albumin as a standard, since this method is less dependent on the amino acid composition of the particular protein. On the basis of this measurement, the purified adenylosuccinate lyase exhibits 1% 280 ) 7.6. Purification of Rabbit Muscle Adenylosuccinate Lyase. Frozen rabbit muscle was thawed at 37 °C for 1 h and cut into small pieces, and the visible fat was removed. The muscle tissue (one box, approximately 800 g) was then homogenized in a Waring blender for 2 min at each speed (slow, medium, and fast) using, for each g of tissue, 4 mL of homogenization buffer [10 mM Tris-HCl buffer, pH 7.4, containing 1 mM EDTA, 0.2 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride (dissolved in 1 mL N,N-dimethylformamide), and 10% glycerol]. The homogenate was centrifuged at 8000 rpm for 1 h, and the supernatant was decanted through eight layers of cheesecloth. The supernatant was brought to 50% ammonium sulfate saturation and was maintained at 4 °C for 6 h. The suspension was centrifuged at 8000 rpm for 1 h, and the resulting supernatant was adjusted to 65% saturation of ammonium sulfate and stood overnight at 4 °C. The precipitate was collected by centrifugation (8000 rpm, 1 h) and dissolved in a minimum volume (∼200 mL) of 10 mM Tris-HCl buffer, pH 7.7, containing 1 mM EDTA, 0.2 mM DTT, and 20% glycerol. The enzyme was then dialyzed extensively against the same buffer. All subsequent purification steps were conducted at room temperature. The dialysate (300 mL) was applied to a
2660 Biochemistry, Vol. 35, No. 8, 1996
FIGURE 2: Electrophoresis of rabbit muscle adenylosuccinate lyase samples in polyacrylamide gels containing SDS after various purification steps. The letters above the lanes refer to the samples (b) after DEAE-cellulose chromatography, (c) after Matrex Blue chromatography, and (d) after FPLC Superose-12 gel filtration.
column of DEAE-cellulose (3 × 40 cm), which was equilibrated with the above buffer. The column was eluted with the same buffer, at a rate of 60 mL/h, until the A280 nm of the eluate was
