Homoserine Dehydrogenase
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Proc. Natl. Acad. Sci. USA Vol. 74, No. 11, pp. 4862-4866, November 1977 Biochemistry Homoserine dehydrogenase: Spontaneous reactivation by dissociation of p-mercuribenzoate from an inactive enzyme-p-mercuribenzoate complex (regulatory enzyme/conformational change/sulfhydryl modification) CAROL CHERKIS EPSTEIN* AND PRASANTA DATTAt Department of Biological Chemistry, The University of Michigan, Ann Arbor, Michigan 48109 Communicated by J. L. Oncley, August 11, 1977 ABSTRACT Incubation of Rhodospirillum rubrum ho- mechanism-dissociation of PMB from an "active-site" -SH moserine dehydrogenase (L-homoserine:NAD+ oxidoreductase, group-and that the rate of reactivation is influenced by the EC 1.1.1.3) with p-mercuribenzoate (PMB) in the presence of various conformational states of the protein. 0.2 M KCI and 2 mM L-threonine resulted in complete loss of enzyme activity. Upon removal of excess PMB, KCI, and L- threonine, a time-dependent recovery of enzyme activity was MATERIALS AND METHODS observed in 25 mM phosphate/i mM EDTA buffer, pH 7.5. Materials. NADP', Tris (Trizma base, highest purity), and Circular dichroism studies indicated that the transition from dithiothreitol (DTT) were from Sigma Chemical Co. 5,5'- inactive to reactivated form of the enzyme was accompanied by a conformational change in the protein. Experiments with Dithiobis(2-nitrobenzoate) (DTNB) was purchased from Cal- [14C]PMB revealed loss of enzyme-bound radioactivity during biochem, and PMB was from Schwarz/Mann. [14C]PMB (10.1 reactivation. Increase in ionic strength of the phosphate buffer mCi/mmol) was purchased from ICN Radiochemicals D)ivision; and/or addition of L-threonine, leading to enzyme aggregation, [14C]toluene standard (4.24 X 105 dpm/ml) and Omnifluor decreased the rate of enzyme reactivation; aggregated enzyme were from New England Nuclear. Amino acids were purchased that remained inactive retained [14C]PMB on the enzyme. from Sigma, Calbiochem, or Schwarz/Mann. All other chem- Sulfhydryl titration of various forms of the enzyme suggested a preferentialrelease of PMB from a sulfhydryl group essential icals were of reagent grade. to enzymic aitivity. We conclude that reactivation of the inac- Enzyme Purification and Assay. Homoserine dehydroge- tive enzyme is due to dissociation of PMB from an "active-site" nase was purified from the photosynthetic bacterium R. rubrum sulfhydryl group and that changes in the protein structure in- SIH (ATCC 25903) according to the method described by Datta fluence the rate of dissociation of the enzyme-PMB complex. (2). An additional Sephadex G-200 gel-filtration step in 0.05 M potassium phosphate buffer, pH 7.5/0.05 M KCl/I mM Homoserine dehydrogenase (L-homoserine:NAD+ oxidore- EDTA/7 mM 2-mercaptoethanol/1 mM DTT was added to ductase, EC 1.1.1.3) of Rhodospirillum rubrum, which cata- this purification scheme to remove a small amount of enzyme lyzes the reversible transformation of aspartate f3-semialdehyde irreversibly aggregated by L-threonine (1). Purity of enzyme and homoserine, has a molecular weight of 110,000 and consists preparations was routinely checked by polyacrylamide gel of two subunits of molecular weight 55,000 (1). A large number electrophoresis as described (2). The procedures for measuring of experiments (1-3) have shown that the enzyme has two -SH enzyme activity in the forward and reverse directions have been groups per 110,000 g, one of which is "buried" in the protein described (2). interior. In the absence of a protein denaturant, the buried -SH Sulfhydryl Titration. Freshly purified enzyme was dialyzed group could be exposed by high concentrations of KC1 or by the exhaustively against buffer S (25 mM potassium phosphate, pH allosteric inhibitor L-threonine. Incubation of the native enzyme 7.5/1 mM EDTA) to remove DTT. Titration of reactive -SH with sulfhydryl reagents did not result in a significant loss of groups with DTNB at 250 was performed by the Ellman catalytic activity; upon addition of L-threonine and KCI to the method (6) as described by Datta (2). A molecular weight of incubation mixture, enzyme inactivation was rapid with a 110,000 (1) was used to calculate the number of -SH groups. half-life of less than 5 min, indicating that the buried -SH group Inactivation of Enzyme and Spontaneous Reactivation. is essential for catalytic activity (1, 3). After exhaustive dialysis against buffer S at 40 to remove DTT, During these studies we observed that, under certain con- purified enzyme was treated at 250 with a 20- to 50-fold molar ditions, enzyme inactivated by p-mercuribenzoate (PMB) in excess of PMB or [14CJPMB in the presence of 2 mM L-threo- buffer containing KCl and threonine was spontaneously reac- nine and 0.2 M KCI until greater than 95% inactivation was tivated when excess PMB, KCI, and threonine were removed achieved. The inactive enzyme was passed through a Sephadex by gel filtration. Similar reactivation of PMB-treated enzyme *G-50 column (0.8 X 56 cm) in buffer S and eluted in 6- to 8-drop has also been reported for pig and beef heart lactate dehydro- fractions at a flow rate of 1 ml/min. Inactive enzyme was lo- genases by Gruber et al. (4) and Massaro and Markert (5), re- cated by assaying fractions for enzyme activity in the presence spectively; in both cases, the process of self-reactivation was of 2.5 mM DTT; peak tubes were pooled and appropriately attributed to the displacement of the organomercurial from treated, and the time course of reactivation was followed by essential to nonessential -SH groups on the enzyme. In this re- assaying enzyme activity in the presence and absence of DTT. port we present data to indicate that reactivation of PMB-in- activated homoserine dehydrogenase occurs by a novel Abbreviations: PMB, p-mercuribenzoate; DTT, dithiothreitol; DTNB, 5,5'-dithiobis(2-nitrobenzoate); t50%, time (hr) to achieve 50% reacti- The costs of publication of this article were defrayed in part by the vation, taken here as reactivation rate; MRW° 208, mean residue el- payment of page charges. This article must therefore be hereby marked lipticity at 208 nm. "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate *Present address: Dow Chemical Company, Midland, MI 48640. this fact. t To whom inquiries should be addressed. 4862 Downloaded by guest on October 1, 2021 Biochemistry: Epstein and Datta Proc. Natl. Acad. Sci. USA 74 (1977) 4863 Table 1. Titration of -SH groups by DTNB* -SH, Enzyme mol/110,000 g z Untreated enzyme 1.50 0 F PMB-inactivated enzymet 0.04 Reactivated enzymel 1.10 C-) OV _ w _ * Titrations were carried out at 250 in buffer S containing 2 mM L- w f threonine and 0.2 M KCl at protein concentrations ranging from l 0.24 to 0.43 mg/mi. ~-. 40 o0 _ t Native enzyme was treated with 35-fold molar excess of PMB in z / buffer S containing 2mM L-threonine plus 0.2 M KCl followed by 0 exhaustive dialysis against buffer S at 4°. 20 Enzyme reactivated 10096 at 250 in buffer S and dialyzed exhaus- 0. tively against buffer S at 4°. o0 the standard. Radioactivity was measured with a cocktail of 0 20 40 60 80 100 120 Omnifluor (4 g/liter of toluene) and Triton X-100, 7:3 (vol/vol), HOURS AT 25° _ _ in_ _ _ _a* 1.TNPackard. Trtiarbe1I1spectrometer model none%3320. AA r["4tytoluene1Ad-ii. I FIG. 1. Kinetics of enzyme reactivation in buffer S. Purified en- standard was used to determine counting efficiency. zyme was inactivated with 25-fold molar excess of PMB and passed through a Sephadex G-50 column as described in Materials and RESULTS Methods. Reactivation in buffer S was followed at 25° at a protein concentration of 375 ug/ml. 0, Assayed with 2.5 mM DTT; 0, assayed Spontaneous Reactivation. The data presented in Fig. 1 without DTT. show that enzyme inactivated by PMB can be fully reactivated by incubation at 250 in buffer S. The kinetics of reactivation In experiments with [14C]PMB, aliquots of reactivating enzyme in buffer S were not significantly different when the enzyme were withdrawn at various times during reactivation and passed was inactivated by incubation with 25-fold molar excess of PMB through a Sephadex G-50 column before duplicate samples of for 15-60 min or by incubation for 30 min with 5- to 110-fold pooled enzyme were taken for protein and radioactivity de- molar excess of PMB. No difference in the reactivation rate was terminations. The results are expressed as mol of [14C]PMB observed when protein concentration during rea'tivation was bound per mol of enzyme. varied from 7.3 to 725 Ag/ml. The optimal pH for reactivation The rate of enzyme reactivationt is expressed as the time (hr) was pH 7.5, and the rates were decreased by about 30% at pH required to achieve 50% reactivation (t50%); enzyme was con- 6.5 and 8.4. The rate of reactivation increased with tempera- sidered to be reactivated 100% when activity regained spon- ture, and at 450 the t5o% value decreased by 5-fold; at 40 no taneously (in the absence of DTT) was equal to that obtained reactivation was observed up to 100 hr. Absence of EDTA in in assay with DTT. Rarely, when enzyme was subjected to ex- buffer S did not influence the inactivation kinetics. It should tremes of pH, salt, or L-threonine, 100% reactivation was not be emphasized that enzyme reactivated once could be re- achieved. From a typical plot of reactivation kinetics (see Fig. peatedly inactivated and reactivated through several cycles. 1), the time required for 50% reactivation was approximately Analogous to the situation reported for the pig heart lactate 50 hr.