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. For eight different enzyme preparations, the t50% values dehydrogenase (4), DTNB-inactivated homoserine dehydro- in buffer S at 250 ranged from 20 to 80 hr; however, most values genase did not reactivate spontaneously. were close to 50 hr. Despite examination of various conditions Dissociation of PMB from Inactive Enzyme. Several to which this variation might be attributed, no satisfactory ex- mechanisms could be envisaged for the spontaneous reactiva- planation has been found. To compare the rates of reactivation tion of an inactive enzyme-PMB complex: (i) transfer of PMB under various experimental conditions, the data are expressed from essential to nonessential -SH groups (4, 5), (ii) a confor- as relative reactivation rate, defined by the to%. in buffer S (run mational change making PMB-modified -SH groups no longer simultaneously with the same enzyme preparation) to that necessary for catalytic activity; and (iii) dissociation of PMB observed under other conditions described for individual ex- from the enzyme. Because the enzyme from R. rubrum con- periments. tains two -SH groups, both of which react with PMB in the Circular Dichroism. Circular dichroism studies were per- presence of KCI and threonine (refs. 1 and 3; see Table 1), it formed according to the general method of Adler et al. (7). A seemed likely that spontaneous reactivation might occur by Jasco Model ord/uv-5 optical rotatory dispersion recorder with dissociation of PMB from the -SH groups on the enzyme. The the Sproul Scientific SS-20 C.D. modification was used; cuvettes data presented in Fig. 2 show that enzyme reactivation was of 0.5 and 1 mm pathlength were used. Calculations of the accompanied by release of enzyme-bound [14C]PMB.§ Control mean residue ellipticity at 208 nm (MRW0 208) for each form experiments revealed that repeated passage of enzyme- of the enzyme were done according to the method of Greenfield [14C]PMB complex through a Sephadex G-50 column soon after and Fasman (8), using the mean residue weight value of 107 obtained from the amino acid content of the enzyme (1) ac- § In several experiments involving modification of both -SH groups cording to the method of Adler et al. (7). on the enzyme with [14C]PMB, values ranging from 1.8 to 3.3 mol Other Methods. Protein concentration was determined by of [14C]PMB per 110,000 g were observed, whereas a value of ap- the method of proximately 2 mol of -SH per 110,000 g was seen with other sulfhy- Lowry et al. (9) with bovine serum albumin as dryl reagents such as DTNB and iodo[14C]acetate in the presence of KCI and threonine or various protein denaturants. It is possible that *Throughout this article the term "rate of enzyme reactivation" is used the hydrophobic nature of the phenyl ring, and the positively charged simply to denote, for comparative purposes, the time taken to re- monovalent mercury, and the negatively charged carboxyl group generate 50% of enzyme activity under various experimental con- may interact with the enzyme to lead to some nonspecific binding ditions; it is not the true rate in the kinetic sense. of [14C]PMB to the enzyme. Downloaded by guest on October 1, 2021 4864 Biochemistry: Epstein and Datta Proc. Nati. Acad. Sci. USA 74 (1977)

3 100 2 6 -i 0 0-so a 80 d

gF 0 aCL _, a:w 40 rIJ -J

z I~6/ m LUeD Inactive i 0W 20 LU I F0 / 0. z W~~~~~~~~~~~~~~~~~~ 0~ LU W~~~~~~~~~~~~~~~~ /

HOURS AT 250 LUa FIG. 2. Kinetics of enzyme reactivation and release of protein- bound [14C]PMB in buffer S. Purified enzyme was inactivated with bydialysis) against-bufferS."Active" 25-fold molar excess of [14C]PMB and passed through Sephadex G-50 -2 column; reactivation was followed at 250 in buffer S. LUX / 0.~ ~ ~~~WVL NGH Reactivatedn inactivation did not release enzyme-bound radioactivity and Li that treatment of [14C]PMB-inactivated enzyme with 2.5 mM whichboth-14 grupNerlble wtndlative[4CP DTT removed almost all radioactivity from the protein with concomitant regeneration of full activity. Titration of -SH groups of PMB-inactivated and fully reac- -16 tivated forms by DTNB in the presence of KC1 and threonine provided additional evidence in this regard (Table 1): the un- 200 210 220 230 240 treated enzyme contained approximately two titratable -SH WAVELENGTH (nm) groups and the inactive enzyme showed no significant reactivity FIG. 3. Circulardichroism spectradofvarious forms oftheenzyme. towards DTNB. Upon spontaneous reactivation of the inactive Native enzyme was dialyzed against buffer S at 400 Inactive enzyme enzyme, approximately 1 mol of -SH per 110,000 g reacted with was prepared by incubation with 41-fold molar excess of PMB fol- DTNB. These results strongly suggest that recovery of enzyme lowed by dialysis at 40 against buffer S. Reactivated enzyme was activity was the consequence of regeneration of a free -SH obtained by spontaneous reactivation of the inactive enzyme followed group on the enzyme. by dialysis against buffer S. "Active" enzyme was prepared by treating Because previous experiments (1-3) had revealed that the native enzyme with 25-fold molar excess of PMB in buffer S alone followed by dialysis against buffer S at 40; 97% of enzyme activity was essential -SH group for catalytic activity was located in the retained. Protein concentrations and cuvette pathlengths were as protein interior and, in the absence of a protein denaturant, follows: native enzyme, 276ag/ml, 1.0 mm; inactive enzyme, 263 could only be made accessible by treating the enzyme with KCI acgIml, 1.0 mm; reactivated enzyme, og/ml,262 0.5 mm; "active" en- and threonine, it was considered likely that spontaneous reac- zyme, 93 jig/ml, 0.5 mm. tivation of the PMB-treated enzyme might be due to loss of PMB from the "inner" -SH group. Several lines of evidence support this notion: (i) whereas titration of the reactivated which both -SH groups were labeled with [e4C]PMB and less enzyme in the presence of KC1 plus threonine showed 1.1 mol than the expected amount of radioactivity (1 mole of [14C]PMB of -SH per 110,000 g, only 0.3 mol/110,000 g was titrated in the per mole of enzyme) remained bound at 100 hr. Some radio- absence of KCl plus threonine, indicating that the "surface" activity was also released when only the "surface" -SH group -SH group still remained complexed with PMB; (ii) when the was blocked by [14C]PMB (see ii above); titration of 0.3 mol of "surface" -SH group reacted with [14C]PMB in the absence of -SH per mol of fully reactivated enzyme in the absence of KC1 KC1 plus threonine (to contain 0.7 mol of [14C]PMB/mol of and threonine (see i above) also supports this notion. enzyme) followed by reaction with nonradioactive PMB in Changes in Protein Conformation. The cumulative data buffer containing KC1 plus threonine to label the "inner" -SH presented thus far indicate that spontaneous reactivation of group, no release of enzyme-bound radioactivity was seen inactive homoserine dehydrogenase occurred by preferential during 80% regeneration of enzyme activity (upon further in- dissociation of PMB from the "inner" -SH group essential to cubation, approximately 30% of radioactivity was released from catalytic activity. It seemed likely, therefore, that large con- the enzyme); and (iii) in reciprocal experiments in which the formational differences in protein structure would distinguish "surface" -SH group was labeled with nonradioactive PMB or the native and inactive enzyme species; upon reactivation, the DTNB and the "inner" -SH with [14C]PMB (1.1 mol of [14C]- native conformation of active enzyme might be expected to PMB/mol of enzyme), progressive release of enzyme-bound reappear. The circular dichroism spectra of native, PMB-in- radioactivity occurred during the entire course of reactivation; activated, and reactivated forms of the enzyme (Fig. 3) indeed approximately 15% of [14C]PMB was retained when the enzyme revealed different conformational states. Whereas the native was fully reactivated. enzyme had a MRWO 208 value- of -13,900, the PMB-inacti- Although the results discussed above clearly show a prefer- vated enzyme showed MRWO 208 = -6,900. The spontaneously ential dissociation of PMB from the "inner" -SH group, upon reactivated enzyme displayed a spectrum with an intermediate prolonged incubation a fraction of the total enzyme molecules MRWO 208 value, -11,300. This apparent discrepancy may be also released their PMB bound to the "surface" -SH group. This explained by the fact that fully reactivated enzyme was not is evident from the data shown in Fig. 2 (also see Fig. 4) in totally free of PMB and was more like a new form of "active" Downloaded by guest on October 1, 2021 Biochemistry: Epstein and Datta Proc. Natl. Acad. Sci. USA 74 (1977) 4865

Li -III I r~~~~~~~~~~~~Table 2. Effects of various ligands on enzyme reactivation* 100 3 j I 0 Relative IX c/ Ligand present reactivation 9 -i during reactivation rate, t50o6t 0 80 20 I\ "I. In buffer S 40 I None 1.00 60 - o I \ ~.J L-Threonine, 2 mM 0.58-0.62 (8) I-- KCl, 0.2 M 0.70 (3) \ I0 2 401- \ a L-Threonine (2 mM) + KCl (0.2 M) 0 (3) I~~~ D-Threonine, 2 mM 1.09 (1) L-Isoleucine, 0.01 M 1.00 (1) w I 21 L-Methionine, 0.01 M 1.11 (1) 20 -I w 0. L-Threonine (2 mM) + L-Isoleucine (0.01 M) 0.95 (2) L-Threonine (2 mM) + L-Methionine (0.01 M) 0.95 (2) KCI (0.2 M) + L-Isoleucine (0.01 vUv c- a M) 0.62 (1) 0 40 80 120 160 200 In modified buffer St HOURS AT 250 None 1.10 (2) FIG. 4. Effects of L-threonine and KCl on reactivation kinetics L-Threonine, 2 mM 1.10 (2) and on release of enzyme-bound [14C]PMB. Enzyme was inactivated * Enzyme was inactivated by incubation with 20- to 50-fold molar by incubation with 21-fold molar excess of [14C]PMB and passed excess ofPMB in buffer S containing 2 mM L-threonine and 0.2 M through a Sephadex G-50 column. Reactivation was followed in buffer KCl and passed through a Sephadex G-50 column equilibrated with S containing 2 mM L-threonine plus 0.2 M KCl up to 100 hr. The the appropriate buffer. The kinetics of reactivation were followed enzyme solution was then dialyzed against buffer S at 40 and enzyme at 250 in the presence of various ligands at protein concentra- reactivation was continued at 250 in buffer S alone. tions ranging from 27 to 177 Ag/ml. t Values in parentheses indicate numbers of experiments done in each enzyme fully functional but with its "surface" -SH group at least case. partially blocked with PMB (cf. Fig. 2 and Table 1). Indeed, 2 mM potassium phosphate, pH 7.5/1 mM EDTA. the spectrum of the native enzyme with only the "surface" -SH group blocked by PMB (indicated by the broken line and counteracted the threonine-mediated retardation of enzyme identified as "active" in Fig. 3) had an MRW0 208 value of reactivation. It is interesting to note that isoleucine was unable -10,900, similar to that of the fully reactivated enzyme. to reverse the retarding effect of KCI on enzyme reactivation. The changes in protein conformation accompanied by This result is not surprising in view of general observations (1, spontaneous reactivation of the inactive enzyme led us to in- 10) that threonine-induced conformational changes are dif- vestigate the kinetics of reactivation in the presence of KCI and ferent from those due to KCI. Control experiments (not shown) threonine, additions that influence the state of aggregation of indicated that glycine and L-alanine, two amino acids not re- the R. rubrum homoserine dehydrogenase (2, 3, 10). The results lated metabolically to homoserine dehydrogenase, did not in- (Fig. 4) show that addition of 0.2 M KCI and 2 mM threonine fluence the rate of reactivation when added to buffer S in the to buffer S prevented enzyme reactivation as well as release of presence or absence of threonine. enzyme-bound [14C]PMB up to a period of 100 hr. When The data in Table 2 also show that in 2 mM potassium threonine and KCI were removed, the same enzyme sample phosphate buffer, pH 7.5, containing 1 mM EDTA, the enzyme showed the usual kinetics of reactivation with concomitant reactivation kinetics were normal; addition of 2 mM L-threo- release of [14C]PMB. We conclude that the altered conforma- nine to this buffer, however, did not retard the rate of enzyme tional state of the enzyme in the presence of KCI and threonine reactivation. This result is consistent with earlier observations did not allow the dissociation of PMB from the "inner" essential (3, 12, 13) that, in buffer low in potassium, the enzyme is de- -SH group so that enzyme reactivation could occur. sensitized to its allosteric L-threonine. Effects of Various Ligands on Reactivation. Because ho- inhibitor, moserine dehydrogenase of R. rubrum is a regulatory enzyme and undergoes various conformational changes in the presence DISCUSSION of several biologically significant metabolites (2, 3, 10), the Numerous examples exist in which the catalytic activity of an effects of these metabolites on spontaneous reactivation were enzyme is abolished by modification of active-site -SH groups examined (Table 2). Addition of 2 mM L-threonine, an allosteric with an organomercurial such as PMB; the enzyme activity may inhibitor, or 0.2 M KCI to buffer S significantly retarded the be restored by treating the inactive enzyme with reducing re- rate of enzyme reactivation; a combination of threonine plus agents to regenerate essential -SH groups. Spontaneous reacti- KCI prevented reactivation. D-Threonine was ineffective. vation of an enzyme inactivated by PMB is an unusual and rare The effects of two other amino acids, L-isoleucine and L- example. Several mechanisms may be proposed to account for methionine, on enzyme reactivation were also examined. These this phenomenon of spontaneous reactivation: (i) transfer of amino acids individually stimulate enzyme activity and, when organomercurial from essential to nonessential -SH groups; (ii) added together with L-threonine, counteract the inhibitory and conformational change that makes blocked -SH groups non- aggregating effects of L-threonine (11). When added separately, essential to enzymic activity, and (iii) dissociation of orga- L-isoleucine and L-methionine had no effect on enzyme reac- nomercurial from the enzyme. Various data (4, 5) on beef and tivation; however, when buffer S was supplemented with 2 mM pig heart lactate dehydrogenases indicate that spontaneous threonine and either 0.01 M isoleucine or 0.01 M methionine, reactivation of PMB-inactivated enzyme is mediated by an the rate of enzyme reactivation was similar to that observed exchange reaction (mechanism i). Because both these enzymes with buffer S alone, indicating that isoleucine and methionine contain a total of 16 -SH groups per mol and only 4 of them are Downloaded by guest on October 1, 2021 4866 Biochemistry: Epstein and Datta Proc. Natl. Acad. Sci. USA 74 (1977) essential to catalytic activity (14, 15), the remaining nonessential Spontaneous reactivation of the R. rubrum homoserine de- -SH groups are generally available for exchange. hydrogenase, a case of dissociation of PMB from a protein -SH Homoserine dehydrogenase of R. rubrum, on the other hand, group, may serve as a model system for investigating mercap- contains two free -SH groups, both of which react with PMB tide bonds in proteins. The ease of dissociation of the mercurial in the presence of KC1 and threonine. This precludes exchange from the enzyme warrants cautious interpretation of experi- as the mechanism of enzyme reactivation. The results of ments in which PMB is used. [14C]PMB binding experiments and the effects of allosteric modifiers on enzyme reactivation kinetics provide direct sup- We thank Dr. R. Zand for his assistance in the circular dichroism port for mechanism iii: conformation-dependent dissociation studies. This work was supported by National Science Foundation of PMB from a -SH group required for enzyme activity. Grant BMS71-00958 and National Institutes of Health Grant GM21436. As yet, the exact temporal relationship between the disso- C.C.E. was a Predoctoral Trainee of the U.S. Public Health Service ciation of PMB and the transition from inactive conformational (Grant GM00187). state to the "active" conformation is not known. It is possible that, as the inactive enzyme-PMB complex undergoes a slow 1. Epstein, C. C. (1976) Ph.D. dissertation, The University of conformational change to the most thermodynamically stable Michigan, Ann Arbor, MI. form, simultaneous release of PMB leads to restoration of en- 2. Datta, P. (1970) J. Biol. Chem. 245,5779-5787. zyme activity. On the other hand, dissociation of PMB could 3. Datta, P. (1971) Biochemistry 10, 402-408. precede the necessary conformational change. A third alter- 4. Gruber, W., Warzecha, K. & Pfleiderer, G. (1962) Biochem. Z. native is that the conformational change to "active" form 336, 107-117. precedes dissociation of PMB; however, with two bulky PMB 5. Massaro, E. J. & Markert, C. L. (1966) Arch. Biochem. Biophys. groups bound to the enzyme, this situation seems unlikely. 116,319-31. Preliminary studies (unpublished data) have revealed an in- 6. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82,70-77. stantaneous release of radioactivity from the enzyme under 7. Adler, A. J., Greenfield, N. J. & Fasman, G. D. (1973) in Methods in Enzymology, eds. Hirs, C. H. W. & Timasheff, S. N. (Academic denaturing conditions, indicating that destruction of the tertiary Press, New York), pp. 675-735. structure removes steric restrictions which, in the native en- 8. Greenfield, N. J. & Fasman, G. D. (1969) Biochemistry 8, zyme, may be rate-limiting for the dissociation to occur. 4108-4116. A cautionary note must be introduced on the significance of 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. the differences in MRWO 208 values between the various forms (1951) J. Biol. Chem. 193,265-275. of homoserine dehydrogenase due to the unknown contribution 10. Datta, P. & Epstein, C. C. (1973) Biochemistry 12, 3888- of the asymmetrically bound residual PMB. Examination of the 3892. circular dichroism spectra of inactive enzyme treated with DTT 11. Datta, P., Dungan, S. M. & Feldberg, R. S. (1973) in Genetics of and of the "active" enzyme with its "surface" -SH group Industrial Microorganisms, eds. Vanek, Z., Hostalek, Z. & blocked with iodoacetate better as to Cudlin, J. (Academia, Prague), pp. 177-193. may provide insight the 12. Datta, P. & Gest, H. (1965) J. Biol. Chem. 240,3023-3033. effect of enzyme-bound PMB on the circular dichroism spec- 13. Mankovitz, R. & Segal, H. L. (1969) Biochemistry 8, 3757- trum. Nevertheless, the known effects of KC1 and the amino 3764. acid modifiers clearly establish that some conformational 14. Pesce, A., McKay, R. H., Stolzenbach, F., Cahn, R. D. & Kaplan, changes occur during the transition from active to inactive N. 0. (1964) J. Biol. Chem. 239, 1753-1761. enzyme and during the subsequent reactivation. 15. Jaenicke, R. & Knof, S. (1968) Eur. J. Biochem. 4, 157-163. Downloaded by guest on October 1, 2021