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Conformational stabilization of enzymes in covalent catalysis
ARCHIVES OF BIOCHEMISTRY Vol. 187, No. 1, April
AND BIOPHYSICS 15. pp. 163-169, 1978
Conformational
Stabilization
MARGUERITE Department
of Biochemistry
of Enzymes
VOLINI’ and Biophysics,
AND
in Covalent
SHU-FANG
University
of Hawaii,
Catalysis’
WANG Honolulu,
Hawaii
96822
Received August 8, 1977; revised December 2, 1977 The enzymes aspartate aminotransferase, rhodanese, and chymotrypsin form covalent substituted-enzyme intermediates during the course of their catalysis. The present analyses show that, in these covalent intermediates, the enzyme proteins are stabilized against pHinduced structural transitions to inert forms that occur in the free enzyme species and other forms not covaiently substituted
The reaction pathways for the three enzymes, aspartate aminotransferase (EC 2.6.1.1), rhodanese (thiosulfate-cyanide sulfurtransferase, EC 2.8.1-l), and chymotrypsin (EC 3.4.4.5). are shown in Fig. 1. All of these enzymes function by double-displacement mechanisms in which covalent substituted-enzyme intermediates are formed (l-8). In each of the pathways as presented, the formation of the covalent intermediate occurs as the second reaction step. For the aminotransferase, which utilizes the coenzyme pyridoxal phosphate, the covalent substituted-enzyme intermediate is the pyridoxal enzyme, E-PLP3 (Fig. la). In the course of this reaction, the pyridoxamine enzyme, E. PMP, combines with the amino-acceptor substrate, a-ketoglutarate, to form an addition complex which decomposes, discharging the product, Z-glutamate, and forming the covalent intermediate (steps 1 and 2). The covalent intermediate subsequently reacts with the amino-donor substrate, I-aspartate, to form a second addition complex which decomposes, discharging the second product, oxalacetate, and regenerating the E. PMP intermediate (steps 3 and 4). By way of contrast, in the E. PMP intermediate the pyridoxamine 1 This work was supported by Research Grant BMS 75-23299 from the National Science Foundation. ’ To whom reprint requests should be addressed. ‘Abbreviations used: E-PLP, pyridozal enzyme; E PMP, pyridozamine enzyme;, E, enzyme; DIP, diisopropylphosphoryk CD, circular dichroism; ORD, optical rotary dispersion; MRW, mean residue weight.
moiety is bound to the apoenzyme in large part by salt linkages. In E-PLP the pyridoxal moiety is bound, in addition, by an aldimine linkage. In the rhodanese-catalyzed reaction the covalent enzyme-sulfur intermediate, E-S, is formed by discharge of the product, sulfite ion, from an addition complex between the free enzyme and the donor substrate, thiosulfate ion (Fig. lb, steps 1 and 2). The acceptor substrate, cyanide ion, combines with the E-S intermediate to form the second product, thiocyanate ion, thereby regenerating the free enzyme. No kinetically significant addition complex is observed with cyanide ion under common experimental conditions (14). With the amino- and sulfurtransferases, the covalent intermediates are isolated by omitting from the reaction mixtures the substrates responsible for their decomposition. Since, in the case of chymotrypsin, this substrate is a water molecule (Fig. lc), other means of producing stable covalent intermediates are commonly used (8-10). In this work the productive covalent intertrimethylacetyl-chymotrypsin, mediate, was selected for study because of its relatively low rate of deacylation (step 3). It is formed by reaction of the free enzyme with the substrate, p-nitrophenyltrimethylacetate (steps 1 and 2). Results with this intermediate were compared with those obtained with the nonproductive covalent intermediate, diisopropylphosphoryl-chymotrypsin. The latter is formed by reaction 163 0003-9861/78/1871-0163$02.00/0 Copyright 0 1978 by Academic Press, Inc. Au rights of reproduction in any form reserved.
164
VOLINI AND WANG
Icompl*x,l
Icomplax,)
I
I
Gl”LEPLPLLASP
Cl bl “+-yz--E’ ISW r-SCN-
0%
1
IE.SSO;i
ho.
w+ - I4 x--E’
pN?-TMA
3
-I-fE.pNP-TMAI IE&
E-rA 31
1. Reaction pathways for (a) aspartate aminotransferase, (b) rhodanese. and (c) chymo-
trypsin. The overall reactions are: (a) a-ketoglutarate + 1-aspsrtate~ (b) SSO3x- + CN-5
Gglutamate + oxalacetate;
SO?- + SCN-; and (c) p-nitrophenyltrimethylacetate + Hz03 triE diisopropylphosphoryl-enzyme
methylacetate + p-nitrophenol or diisopropyltluorophosphate + F-.
of the free enzyme with the irreversible inhibitor, diisopropyh’iuorophosphate (steps 1 and 2). StructuraI studies related to those reported here have been described previously with these chymotrypsin intermediates (8-10). In the present report, the conformational stabilities to changes in pH are examined for ah of these covalent enzyme intermediates. They are compared with those for the enzyme forms indicated in Fig. 1, which undergo structural transitions to inert conformers designated as E’. MATERIALS
AND METHODS
Enzyme preparations. Aspartate aminotransferase wss purchased from the Sigma Chemical Co. as a suspension containii 3 M ammonium sulfate, 50 nnu maleate, and 2.5 mu a-ketoglutarate. Ten milliliters of the suspension was centrifuged. The pellet was dissolved in 4 ml of 0.5 mM sodium acetate buffer. It was subsequently dialyzed against two l-liter changes of 0.05 rnr+rsodium acetate buffer, pH 5.8. For most of the spectral experiments, 0.05ml aliquots of this solution were diluted either with 0.3 ml of Tris-acetate buffer, pH 8.3, 30 mM in acetate, or with 0.3 ml of 10 mM sodium acetate, pH 4.0. The stock enzyme solution was assayed before and after spectra were recorded. Except for specific activity measurements, the assay mixture contained 1 ml of Tris-acetate buffer (pH 8.3,
ionic strength 0.4), 30 pmol of a-ketoglutsrate (neutralized), 60 amol of Caspartate (neutralized), in a tinal volume of 3.0 ml. For specific activity measurements, the reaction mixtures were the same as specified by Sizer and Jenkins in Method II (11). Rates were measured by following the appearance of oxalacetate absorbance at 280 nm. Protein concentrations were estimated by a biuret method (12). The specific activity at 37% was 2.9 U/min/mg, according to Method II reported by Sizer and Jenkins (11). Crystalline beef liver rhodanese was prepared by the method of Horowitz and De Toma (13) with the modifications described previously (14). Protein and activity measurements were the same as given previously (14). For spectral measurements, the stock protein solution was diluted with either Na-K phosphate buffer, pH 7.0, or Tris-sulfate buffer (ionic strength 0.1, pH 8.6). Crystalline a-chymotrypsin and diiiopropylphosphoryl (DIP)-chymotrypsin were obtained from the Worthington Corp. Trimethylacetyl chymotrypsin wss prepared from a-chymotrypsin and recrystallixed p-nitrotrimethylacetate. Stock solutions in 0.1 M KC1 at pH 3.55 were diluted with Tris-acetate buffers, 0.1 ionic strength, for the spectral measurements. Protein concentrations and enzyme activity were measured as described previously (10). Spectral determinations. Circular dichroism (CD) and optical rotatory dispersion (ORD) spectra were recorded on a Cary Model 60 spectropolarimeter with CD attachment. Absorption spectra were recorded on a Cary Model 15 spectrophotometer. For the amino-
CONFORMATIONAL
STABILIZATION
transferase, protein concentrations varied from 1.0 to 2.0 mg/ml. For rhodanese and chymotrypsin, the protein concentration was 0.9 mg/ml. The samples were examined in cells of O.l- or 0.5~mm path length at a sensitivity of 0.1 or 0.04” full scale. The ellipticity values (@MRW for rhodanese and chymotrypsin were calculated using a mean residue weight of 115. The reduced molar rotation values (m’)uaw for the aminotransferase were corrected for solvent refractive index. They are based on a mean residue weight of 114 (15). RESULTS
Aspartate aminotransferase. The sequence shown in Fig. la was considered as two half-reactions at equilibrium. The relative concentrations of the pyridoxal and pyridoxamine intermediates, E-PLP and E. PMP, were varied by adding different concentrations of cy-ketoglutarate and dlglutamate to solutions of the enzyme initially in the E-PLP form. In the diagram, (complexr) is meant to represent both the (E-PLP . GLU) complex and its isomerized form (E . PMP .~YKG). As pointed out by Velick and Vavra (l), at low substrate concentrations there is no appreciable accumulation of the complexes, and the ratio of the intermediates is given by the expression. K=KIKz=
(Glu) (E - PLP) ((w- KG) (E.PMP) ’
where KI and KZ are the equilibrium constants for steps 1 and 2 in Fig. la. At pH 8.3 the relative concentrations of the two intermediates were estimated, using as a guide the values of the constants determined kinetically by Velick and Vavra (1) and Henson and Cleland (2). In Fig. 2 (right), the ORD spectrum of the enzyme treated with a-ketoglutarate at pH 8.3 is compared with that of the enzyme treated with a-ketoglutarate at pH 4.0. As shown, no significant difference was observed, indicating that the conformation of the covalent pyridoxal intermediate is unchanged by the alteration in pH. The same conformation was observed with untreated enzyme solutions, in accordance with the fact that the enzyme was initially in the pyridoxal form. The ORD spectrum of the enzyme treated with &glutamate at pH 8.3 (Fig. 2,
165
OF ENZYMES
left) was essentially the same as that observed for the enzyme treated with cr-ketoglutarate. However, upon treatment with d&glutamate in concentrations of 1.4-4.2 mu at pH 4.0, decreases in the reduced molar rotation to 3900 deg-cm2 dmol-’ were observed at 199 nm (Fig. 2, left). This represents a 14% change in the rotation observed at pH 8.3. Since the experimental error was <3%, the difference indicates that there is a major structural transition in the protein upon formation of the noncovalently substituted pyridoxamine intermediate at the low pH. To avoid corrections for the optical activity of the substrate, d&glutamate was used. The rotation values of the I-enantiomorph were examined separately. From these values, it was clear that the correction was negligible for uptake of the Z-isomer under the experimental conditions. The experiments were repeated using somewhat different protein concentrations from 1.0 to 2.0 mg/ml. The results of all the experiments were in agreement. Rhodanese. In parallel with the aminotransferase, the rhodanese reaction was considered as two half-reactions at equilibrium (Fig. lb). The free enzyme form, E, and the enzyme-sulfur intermediate, E-S, were generated either by isolating the enzyme in these forms or by estimating their relative concentrations upon the addition of varying concentrations of thiosulfate and sulfite ions. In the latter case, the ratio of the intermediates was estimated from the expression. K =
KlK2
(E) @S032-) = (E-S) (so32-)
7
where the values of the equilibrium constants were those reported previously (14). In Fig. 3 (right), the CD spectrum of the covalent enzyme-sulfur intermediate in the wavelength region from 250 to 202 nm is compared at pH 8.6 and pH 7.0. The spectrum is essentially unchanged by the alteration in PH. In Fig. 3 (left), the difference in the CD spectra of the free enzyme form upon the same change in pH indicates that there is a major structural alteration in the free enzyme at pH 7.0. That this conformer is
166
VOLINI
AND
WANG
I.mL
1 200
220
240
200
260
.
.
220
240
260
Xnm
FIG. 2. ORD spectra of aspartate aminotransferase. Upper right: enzyme with a-ketoglutarate, and pH 4.0 (M). Lower right: difference spectrum. Upper left: 0.67 mM, at pH 8.3 (A -A) enzyme with &glutamate, 4.2 mM, at pH 8.3 (A-A) and pH 4.0 (u). Lower left: difference spectrum.
inactive is in accordance with the results of kinetic studies which show that the rate decreases by close to two orders of magnitude at pH 7.0 (5, 16). The difference in molar ellipticity (MRW) is close to 2000 deg-cm’ dmol-’ at 208 nm, or 22% of the value observed at pH 8.6 for the active conformer. Chymotrypsin. Trimethylacetyl chymotrypsin is isolated at acidic pH where the rate of deacylation is negligible. At alkaline pH, where the deacylation rate is relatively high, the spectrum of the covalent trimethylacetyl intermediate was approximated by extrapolation of ellipticity values near the extrema to zero time. It was shown previously that the change in the negative extremum near 228 nm at pH 9.0 is a firstorder reaction and correlates with the rate
of deacylation (10). Further, the ellipticity value at zero time for the E-TMA intermediate was close to that of the DIP intermediate which does not undergo deacylation (Fig. lc). In Fig. 4 (right), the CD spectrum for the DIP intermediate from 250 to 220 nm is compared at pH 3.8 and pH 9.0. No significant difference is observed for the two intermediates. From the agreement in the values near the extremum it is reasonable to presume that these spectra follow closely those for the covalent E-TMA intermediate under the same conditions. In Fig. 4 (left), the CD spectra are shown for the free enzyme under the same conditions. The difference in molar ellipticity values (MRW) at 228 nm is 1000 deg-cm2 dmol-‘, or 28% of the value observed at pH
CONFORMATIONAL
STABILIZATION
167
OF ENZYMES
l
n .
I 210
230
I
mm’
,
25
210
230
25C
x nm FIG. 3. CD spectra of rhodanese. Upper right: The enzyme-sulfur intermediate, E-S, was generated by adding 0.30 mu SSO?, at pH 8.6 (A-A) and at pH 7.0 (u). Lower right: difference spectrum. Upper left: The free enzyme, E, was generated by adding 0.01 mM SSOa*and 0.20 mM SO?, at pH 8.6 (A-A) and at pH 7.0 (u). Lower left: difference spectrum.
3.8. In contrast to the results given above for the amino- and sulfurtransferases, it is the deprotonated form of chymotrypsin that is observed as the inert conformer in these experiments (8). DISCUSSION
The results of these studies show that, for three enzymes that function by covalent catalysis (an aminotransferase, a sulfurtransferase, and a hydrolase), the covalent intermediates are stabilized against pH-induced structural transitions. Previous rate studies indicate that the structural transitions observed in noncovalent intermediates of the three enzymes upon alteration of the solvent pH are conversions to catalytically inert conformers.
CD and ORD spectra of the inert conformers in the wavelength region of peptide chromophore absorption evidence spectral changes ranging from 14 to 28% of the rotation or ellipticity values observed for the active enzyme forms. The difference values are calculated on a mean residue weight basis, and as such are averaged over all peptide chromophores in the enzyme molecules. The magnitudes of the differences when based on the molecular weights of the individual enzymes make it clear that the changes with the transferases result from a spatial rearrangement of several amino acid residues in the protein. As operationally defined here, these are major structural changes. This definition serves to distinguish such changes from structural altera-
168
VOLINI
AND
WANG
. .
-
n
I. -
l
.
FIG. 4. CD spectra of chymotrypsin. Upper right: DIP intermediate, at pH 3.8 (A-A) and at pH 9.0 (M). The TMA intermediate shows no significant difference at acid pH, and gives the same ellipticity values near the extremum at pH 9.0. Lower right: difference spectrum. Upper left: the free enzyme, at pH 3.8 (A-A) and at pH 9.0 (M). Lower left: difference spectrum.
tions which involve only one or two residues or slight differences in bond lengths.4 For example, with the aminotransferase, the difference in rotation near 200 nm based on the molecular weight of the protein is 1.7 x lo6 deg-cm2 dmol-‘. This may be compared with the reduced molar rotation per residue near 200 nm in the u-helical conformer of polylysine (7.5 x 104deg-cm2 dmol-‘) (17). The observed change is some 20-fold larger than this value. It is some 50fold larger than the value of the /I conformer of polylysine (17). By comparison with known compounds, this change is far too large to result from vi&al perturbations by a bound glutamate or pyridoxamine moiety. The inactivation of the aminotransferase which has been observed below pH 5.0 (1) can be attributed in p,art to the formation of the destabilized conformer upon conversion of the covalent phospho4 The magnitude of these major changes may still be small when compared to those undergone by the protein when it converts to a completely unfolded state.
pyridoxal intermediate to the pyridoxamine form during the course of the initial turnover cycles. Rate studies with rhodanese indicate that, at pH 7.0, the sulfurtransferase activity is decreased by almost two orders of magnitude from that observed at pH 8.6 (16). Together with this observation, the CD measurements indicate that there is conversion of the free enzyme species to an inert conformer, whereas a structural change does not occur in the covalent enzyme-sulfur intermediate under the same conditions. The present results show that the conformational stability observed in previous work with the nonproductive DIP intermediate of chymotrypsin at alkaline pH (8) is also observed with the productive trimethylacetyl intermediate. The change in ellipticity observed with free chymotrypsin at alkaline pH is smaller than that observed with the transferases, but sufficient to make the enzyme inactive (8). This change, as pointed out by McCann et al. (8), most
CONFORMATIONAL
STABILIZATION
likely is a consequence of deprotonation of the a-amino group of the N-terminal isoleutine residue resulting in dissolution of an ion pair with an aapartate residue. With the transferases, the causes of the structural changes are still unclear. For each enzyme, the fraction of molecules present in the form susceptible to conformational inactivation is determined by the ratio of concentrations of donor and acceptor substrates and the concentrations of products. These factors may operate similarly in uiuo to control the fraction of catalytically competent enzyme molecules. REFERENCES 1. VELICK, S. F., AND VAVRA, J. (1982) J..Biol. Chem. 237,2109-2122. 2. HENSON, C. P., AND &ELAND, W. W. (1964) Bb
chemistry 3,338-345. 3. JENKINS, W. T., AND SIZER, I. W. (1959) J. Biol.
Chem. 236,620-623. 4. WESTLEY, J. (1973) Advan. Enzymol. 39,327-368. 5. VOLINI, M., AND WESTLEY, J. (1966) J. Biol. Chem. 241,5166-5176. 6. MCCONN, J., Ku, E., ODELL, C., AND CZERLINKSI, G. (1988) Science 161.274-276. 7. BENDER, M. L., AND KEZDY, F. J. (1965) Annu.
OF ENZYMES
169
Rev. Biochem. 34,49-76. 8. MCCONN, J., FASMAN, G. D., AND HESS, G. (1989)
J. Mol. Biol. 39,551~562. 9. BILTONEN, R., LUMRY, R., MADISON, V., AND PARKER, H. (1985) Proc. Nat. Acad. Sci. USA S4,1016-1025. 10. VOLINI, M., AND TOBIAS, P. (1969) J. Biol. Chem. 244,5105-5109. 11. SIZER, I. W., AND JENKINS, W. F. (1962) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 5, pp. 677-684, Academic Press, New York. 12. ZAMENHOF, S. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 3, p. 702, Academic Press, New York. 13. HOROWITZ, P., AND DE TOMA, F. (1970) J. Biol.
Chem. 246,984-985. 14. WANG, S. F., AND VOLINI, M. (1973) J. Biol. Chem. 248, 7376-7365. 15. MARTINEZ-CARRION, M., TIEMEIER, D. C., AND PETERSON, D. L. (1970) Biochemistry 9, 2574-2562. 16. SCHLESINGER, P., AND WESTLEY, J. (1974) J. Biol.
Chem. 249,780-788. 17. TIMASHEFF, S. N., Susr, H., TOWNEND, R., STEVENS, L., GORBUNOFF, M. J., AND KUMOSINSKI, T. F. (1967) in Conformation of Biopolymera (Ramachandran, G. N., ed.), Vol. 1, p. 173, Academic Press, London.
AND BIOPHYSICS 15. pp. 163-169, 1978
Conformational
Stabilization
MARGUERITE Department
of Biochemistry
of Enzymes
VOLINI’ and Biophysics,
AND
in Covalent
SHU-FANG
University
of Hawaii,
Catalysis’
WANG Honolulu,
Hawaii
96822
Received August 8, 1977; revised December 2, 1977 The enzymes aspartate aminotransferase, rhodanese, and chymotrypsin form covalent substituted-enzyme intermediates during the course of their catalysis. The present analyses show that, in these covalent intermediates, the enzyme proteins are stabilized against pHinduced structural transitions to inert forms that occur in the free enzyme species and other forms not covaiently substituted
The reaction pathways for the three enzymes, aspartate aminotransferase (EC 2.6.1.1), rhodanese (thiosulfate-cyanide sulfurtransferase, EC 2.8.1-l), and chymotrypsin (EC 3.4.4.5). are shown in Fig. 1. All of these enzymes function by double-displacement mechanisms in which covalent substituted-enzyme intermediates are formed (l-8). In each of the pathways as presented, the formation of the covalent intermediate occurs as the second reaction step. For the aminotransferase, which utilizes the coenzyme pyridoxal phosphate, the covalent substituted-enzyme intermediate is the pyridoxal enzyme, E-PLP3 (Fig. la). In the course of this reaction, the pyridoxamine enzyme, E. PMP, combines with the amino-acceptor substrate, a-ketoglutarate, to form an addition complex which decomposes, discharging the product, Z-glutamate, and forming the covalent intermediate (steps 1 and 2). The covalent intermediate subsequently reacts with the amino-donor substrate, I-aspartate, to form a second addition complex which decomposes, discharging the second product, oxalacetate, and regenerating the E. PMP intermediate (steps 3 and 4). By way of contrast, in the E. PMP intermediate the pyridoxamine 1 This work was supported by Research Grant BMS 75-23299 from the National Science Foundation. ’ To whom reprint requests should be addressed. ‘Abbreviations used: E-PLP, pyridozal enzyme; E PMP, pyridozamine enzyme;, E, enzyme; DIP, diisopropylphosphoryk CD, circular dichroism; ORD, optical rotary dispersion; MRW, mean residue weight.
moiety is bound to the apoenzyme in large part by salt linkages. In E-PLP the pyridoxal moiety is bound, in addition, by an aldimine linkage. In the rhodanese-catalyzed reaction the covalent enzyme-sulfur intermediate, E-S, is formed by discharge of the product, sulfite ion, from an addition complex between the free enzyme and the donor substrate, thiosulfate ion (Fig. lb, steps 1 and 2). The acceptor substrate, cyanide ion, combines with the E-S intermediate to form the second product, thiocyanate ion, thereby regenerating the free enzyme. No kinetically significant addition complex is observed with cyanide ion under common experimental conditions (14). With the amino- and sulfurtransferases, the covalent intermediates are isolated by omitting from the reaction mixtures the substrates responsible for their decomposition. Since, in the case of chymotrypsin, this substrate is a water molecule (Fig. lc), other means of producing stable covalent intermediates are commonly used (8-10). In this work the productive covalent intertrimethylacetyl-chymotrypsin, mediate, was selected for study because of its relatively low rate of deacylation (step 3). It is formed by reaction of the free enzyme with the substrate, p-nitrophenyltrimethylacetate (steps 1 and 2). Results with this intermediate were compared with those obtained with the nonproductive covalent intermediate, diisopropylphosphoryl-chymotrypsin. The latter is formed by reaction 163 0003-9861/78/1871-0163$02.00/0 Copyright 0 1978 by Academic Press, Inc. Au rights of reproduction in any form reserved.
164
VOLINI AND WANG
Icompl*x,l
Icomplax,)
I
I
Gl”LEPLPLLASP
Cl bl “+-yz--E’ ISW r-SCN-
0%
1
IE.SSO;i
ho.
w+ - I4 x--E’
pN?-TMA
3
-I-fE.pNP-TMAI IE&
E-rA 31
1. Reaction pathways for (a) aspartate aminotransferase, (b) rhodanese. and (c) chymo-
trypsin. The overall reactions are: (a) a-ketoglutarate + 1-aspsrtate~ (b) SSO3x- + CN-5
Gglutamate + oxalacetate;
SO?- + SCN-; and (c) p-nitrophenyltrimethylacetate + Hz03 triE diisopropylphosphoryl-enzyme
methylacetate + p-nitrophenol or diisopropyltluorophosphate + F-.
of the free enzyme with the irreversible inhibitor, diisopropyh’iuorophosphate (steps 1 and 2). StructuraI studies related to those reported here have been described previously with these chymotrypsin intermediates (8-10). In the present report, the conformational stabilities to changes in pH are examined for ah of these covalent enzyme intermediates. They are compared with those for the enzyme forms indicated in Fig. 1, which undergo structural transitions to inert conformers designated as E’. MATERIALS
AND METHODS
Enzyme preparations. Aspartate aminotransferase wss purchased from the Sigma Chemical Co. as a suspension containii 3 M ammonium sulfate, 50 nnu maleate, and 2.5 mu a-ketoglutarate. Ten milliliters of the suspension was centrifuged. The pellet was dissolved in 4 ml of 0.5 mM sodium acetate buffer. It was subsequently dialyzed against two l-liter changes of 0.05 rnr+rsodium acetate buffer, pH 5.8. For most of the spectral experiments, 0.05ml aliquots of this solution were diluted either with 0.3 ml of Tris-acetate buffer, pH 8.3, 30 mM in acetate, or with 0.3 ml of 10 mM sodium acetate, pH 4.0. The stock enzyme solution was assayed before and after spectra were recorded. Except for specific activity measurements, the assay mixture contained 1 ml of Tris-acetate buffer (pH 8.3,
ionic strength 0.4), 30 pmol of a-ketoglutsrate (neutralized), 60 amol of Caspartate (neutralized), in a tinal volume of 3.0 ml. For specific activity measurements, the reaction mixtures were the same as specified by Sizer and Jenkins in Method II (11). Rates were measured by following the appearance of oxalacetate absorbance at 280 nm. Protein concentrations were estimated by a biuret method (12). The specific activity at 37% was 2.9 U/min/mg, according to Method II reported by Sizer and Jenkins (11). Crystalline beef liver rhodanese was prepared by the method of Horowitz and De Toma (13) with the modifications described previously (14). Protein and activity measurements were the same as given previously (14). For spectral measurements, the stock protein solution was diluted with either Na-K phosphate buffer, pH 7.0, or Tris-sulfate buffer (ionic strength 0.1, pH 8.6). Crystalline a-chymotrypsin and diiiopropylphosphoryl (DIP)-chymotrypsin were obtained from the Worthington Corp. Trimethylacetyl chymotrypsin wss prepared from a-chymotrypsin and recrystallixed p-nitrotrimethylacetate. Stock solutions in 0.1 M KC1 at pH 3.55 were diluted with Tris-acetate buffers, 0.1 ionic strength, for the spectral measurements. Protein concentrations and enzyme activity were measured as described previously (10). Spectral determinations. Circular dichroism (CD) and optical rotatory dispersion (ORD) spectra were recorded on a Cary Model 60 spectropolarimeter with CD attachment. Absorption spectra were recorded on a Cary Model 15 spectrophotometer. For the amino-
CONFORMATIONAL
STABILIZATION
transferase, protein concentrations varied from 1.0 to 2.0 mg/ml. For rhodanese and chymotrypsin, the protein concentration was 0.9 mg/ml. The samples were examined in cells of O.l- or 0.5~mm path length at a sensitivity of 0.1 or 0.04” full scale. The ellipticity values (@MRW for rhodanese and chymotrypsin were calculated using a mean residue weight of 115. The reduced molar rotation values (m’)uaw for the aminotransferase were corrected for solvent refractive index. They are based on a mean residue weight of 114 (15). RESULTS
Aspartate aminotransferase. The sequence shown in Fig. la was considered as two half-reactions at equilibrium. The relative concentrations of the pyridoxal and pyridoxamine intermediates, E-PLP and E. PMP, were varied by adding different concentrations of cy-ketoglutarate and dlglutamate to solutions of the enzyme initially in the E-PLP form. In the diagram, (complexr) is meant to represent both the (E-PLP . GLU) complex and its isomerized form (E . PMP .~YKG). As pointed out by Velick and Vavra (l), at low substrate concentrations there is no appreciable accumulation of the complexes, and the ratio of the intermediates is given by the expression. K=KIKz=
(Glu) (E - PLP) ((w- KG) (E.PMP) ’
where KI and KZ are the equilibrium constants for steps 1 and 2 in Fig. la. At pH 8.3 the relative concentrations of the two intermediates were estimated, using as a guide the values of the constants determined kinetically by Velick and Vavra (1) and Henson and Cleland (2). In Fig. 2 (right), the ORD spectrum of the enzyme treated with a-ketoglutarate at pH 8.3 is compared with that of the enzyme treated with a-ketoglutarate at pH 4.0. As shown, no significant difference was observed, indicating that the conformation of the covalent pyridoxal intermediate is unchanged by the alteration in pH. The same conformation was observed with untreated enzyme solutions, in accordance with the fact that the enzyme was initially in the pyridoxal form. The ORD spectrum of the enzyme treated with &glutamate at pH 8.3 (Fig. 2,
165
OF ENZYMES
left) was essentially the same as that observed for the enzyme treated with cr-ketoglutarate. However, upon treatment with d&glutamate in concentrations of 1.4-4.2 mu at pH 4.0, decreases in the reduced molar rotation to 3900 deg-cm2 dmol-’ were observed at 199 nm (Fig. 2, left). This represents a 14% change in the rotation observed at pH 8.3. Since the experimental error was <3%, the difference indicates that there is a major structural transition in the protein upon formation of the noncovalently substituted pyridoxamine intermediate at the low pH. To avoid corrections for the optical activity of the substrate, d&glutamate was used. The rotation values of the I-enantiomorph were examined separately. From these values, it was clear that the correction was negligible for uptake of the Z-isomer under the experimental conditions. The experiments were repeated using somewhat different protein concentrations from 1.0 to 2.0 mg/ml. The results of all the experiments were in agreement. Rhodanese. In parallel with the aminotransferase, the rhodanese reaction was considered as two half-reactions at equilibrium (Fig. lb). The free enzyme form, E, and the enzyme-sulfur intermediate, E-S, were generated either by isolating the enzyme in these forms or by estimating their relative concentrations upon the addition of varying concentrations of thiosulfate and sulfite ions. In the latter case, the ratio of the intermediates was estimated from the expression. K =
KlK2
(E) @S032-) = (E-S) (so32-)
7
where the values of the equilibrium constants were those reported previously (14). In Fig. 3 (right), the CD spectrum of the covalent enzyme-sulfur intermediate in the wavelength region from 250 to 202 nm is compared at pH 8.6 and pH 7.0. The spectrum is essentially unchanged by the alteration in PH. In Fig. 3 (left), the difference in the CD spectra of the free enzyme form upon the same change in pH indicates that there is a major structural alteration in the free enzyme at pH 7.0. That this conformer is
166
VOLINI
AND
WANG
I.mL
1 200
220
240
200
260
.
.
220
240
260
Xnm
FIG. 2. ORD spectra of aspartate aminotransferase. Upper right: enzyme with a-ketoglutarate, and pH 4.0 (M). Lower right: difference spectrum. Upper left: 0.67 mM, at pH 8.3 (A -A) enzyme with &glutamate, 4.2 mM, at pH 8.3 (A-A) and pH 4.0 (u). Lower left: difference spectrum.
inactive is in accordance with the results of kinetic studies which show that the rate decreases by close to two orders of magnitude at pH 7.0 (5, 16). The difference in molar ellipticity (MRW) is close to 2000 deg-cm’ dmol-’ at 208 nm, or 22% of the value observed at pH 8.6 for the active conformer. Chymotrypsin. Trimethylacetyl chymotrypsin is isolated at acidic pH where the rate of deacylation is negligible. At alkaline pH, where the deacylation rate is relatively high, the spectrum of the covalent trimethylacetyl intermediate was approximated by extrapolation of ellipticity values near the extrema to zero time. It was shown previously that the change in the negative extremum near 228 nm at pH 9.0 is a firstorder reaction and correlates with the rate
of deacylation (10). Further, the ellipticity value at zero time for the E-TMA intermediate was close to that of the DIP intermediate which does not undergo deacylation (Fig. lc). In Fig. 4 (right), the CD spectrum for the DIP intermediate from 250 to 220 nm is compared at pH 3.8 and pH 9.0. No significant difference is observed for the two intermediates. From the agreement in the values near the extremum it is reasonable to presume that these spectra follow closely those for the covalent E-TMA intermediate under the same conditions. In Fig. 4 (left), the CD spectra are shown for the free enzyme under the same conditions. The difference in molar ellipticity values (MRW) at 228 nm is 1000 deg-cm2 dmol-‘, or 28% of the value observed at pH
CONFORMATIONAL
STABILIZATION
167
OF ENZYMES
l
n .
I 210
230
I
mm’
,
25
210
230
25C
x nm FIG. 3. CD spectra of rhodanese. Upper right: The enzyme-sulfur intermediate, E-S, was generated by adding 0.30 mu SSO?, at pH 8.6 (A-A) and at pH 7.0 (u). Lower right: difference spectrum. Upper left: The free enzyme, E, was generated by adding 0.01 mM SSOa*and 0.20 mM SO?, at pH 8.6 (A-A) and at pH 7.0 (u). Lower left: difference spectrum.
3.8. In contrast to the results given above for the amino- and sulfurtransferases, it is the deprotonated form of chymotrypsin that is observed as the inert conformer in these experiments (8). DISCUSSION
The results of these studies show that, for three enzymes that function by covalent catalysis (an aminotransferase, a sulfurtransferase, and a hydrolase), the covalent intermediates are stabilized against pH-induced structural transitions. Previous rate studies indicate that the structural transitions observed in noncovalent intermediates of the three enzymes upon alteration of the solvent pH are conversions to catalytically inert conformers.
CD and ORD spectra of the inert conformers in the wavelength region of peptide chromophore absorption evidence spectral changes ranging from 14 to 28% of the rotation or ellipticity values observed for the active enzyme forms. The difference values are calculated on a mean residue weight basis, and as such are averaged over all peptide chromophores in the enzyme molecules. The magnitudes of the differences when based on the molecular weights of the individual enzymes make it clear that the changes with the transferases result from a spatial rearrangement of several amino acid residues in the protein. As operationally defined here, these are major structural changes. This definition serves to distinguish such changes from structural altera-
168
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AND
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-
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.
FIG. 4. CD spectra of chymotrypsin. Upper right: DIP intermediate, at pH 3.8 (A-A) and at pH 9.0 (M). The TMA intermediate shows no significant difference at acid pH, and gives the same ellipticity values near the extremum at pH 9.0. Lower right: difference spectrum. Upper left: the free enzyme, at pH 3.8 (A-A) and at pH 9.0 (M). Lower left: difference spectrum.
tions which involve only one or two residues or slight differences in bond lengths.4 For example, with the aminotransferase, the difference in rotation near 200 nm based on the molecular weight of the protein is 1.7 x lo6 deg-cm2 dmol-‘. This may be compared with the reduced molar rotation per residue near 200 nm in the u-helical conformer of polylysine (7.5 x 104deg-cm2 dmol-‘) (17). The observed change is some 20-fold larger than this value. It is some 50fold larger than the value of the /I conformer of polylysine (17). By comparison with known compounds, this change is far too large to result from vi&al perturbations by a bound glutamate or pyridoxamine moiety. The inactivation of the aminotransferase which has been observed below pH 5.0 (1) can be attributed in p,art to the formation of the destabilized conformer upon conversion of the covalent phospho4 The magnitude of these major changes may still be small when compared to those undergone by the protein when it converts to a completely unfolded state.
pyridoxal intermediate to the pyridoxamine form during the course of the initial turnover cycles. Rate studies with rhodanese indicate that, at pH 7.0, the sulfurtransferase activity is decreased by almost two orders of magnitude from that observed at pH 8.6 (16). Together with this observation, the CD measurements indicate that there is conversion of the free enzyme species to an inert conformer, whereas a structural change does not occur in the covalent enzyme-sulfur intermediate under the same conditions. The present results show that the conformational stability observed in previous work with the nonproductive DIP intermediate of chymotrypsin at alkaline pH (8) is also observed with the productive trimethylacetyl intermediate. The change in ellipticity observed with free chymotrypsin at alkaline pH is smaller than that observed with the transferases, but sufficient to make the enzyme inactive (8). This change, as pointed out by McCann et al. (8), most
CONFORMATIONAL
STABILIZATION
likely is a consequence of deprotonation of the a-amino group of the N-terminal isoleutine residue resulting in dissolution of an ion pair with an aapartate residue. With the transferases, the causes of the structural changes are still unclear. For each enzyme, the fraction of molecules present in the form susceptible to conformational inactivation is determined by the ratio of concentrations of donor and acceptor substrates and the concentrations of products. These factors may operate similarly in uiuo to control the fraction of catalytically competent enzyme molecules. REFERENCES 1. VELICK, S. F., AND VAVRA, J. (1982) J..Biol. Chem. 237,2109-2122. 2. HENSON, C. P., AND &ELAND, W. W. (1964) Bb
chemistry 3,338-345. 3. JENKINS, W. T., AND SIZER, I. W. (1959) J. Biol.
Chem. 236,620-623. 4. WESTLEY, J. (1973) Advan. Enzymol. 39,327-368. 5. VOLINI, M., AND WESTLEY, J. (1966) J. Biol. Chem. 241,5166-5176. 6. MCCONN, J., Ku, E., ODELL, C., AND CZERLINKSI, G. (1988) Science 161.274-276. 7. BENDER, M. L., AND KEZDY, F. J. (1965) Annu.
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Rev. Biochem. 34,49-76. 8. MCCONN, J., FASMAN, G. D., AND HESS, G. (1989)
J. Mol. Biol. 39,551~562. 9. BILTONEN, R., LUMRY, R., MADISON, V., AND PARKER, H. (1985) Proc. Nat. Acad. Sci. USA S4,1016-1025. 10. VOLINI, M., AND TOBIAS, P. (1969) J. Biol. Chem. 244,5105-5109. 11. SIZER, I. W., AND JENKINS, W. F. (1962) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 5, pp. 677-684, Academic Press, New York. 12. ZAMENHOF, S. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 3, p. 702, Academic Press, New York. 13. HOROWITZ, P., AND DE TOMA, F. (1970) J. Biol.
Chem. 246,984-985. 14. WANG, S. F., AND VOLINI, M. (1973) J. Biol. Chem. 248, 7376-7365. 15. MARTINEZ-CARRION, M., TIEMEIER, D. C., AND PETERSON, D. L. (1970) Biochemistry 9, 2574-2562. 16. SCHLESINGER, P., AND WESTLEY, J. (1974) J. Biol.
Chem. 249,780-788. 17. TIMASHEFF, S. N., Susr, H., TOWNEND, R., STEVENS, L., GORBUNOFF, M. J., AND KUMOSINSKI, T. F. (1967) in Conformation of Biopolymera (Ramachandran, G. N., ed.), Vol. 1, p. 173, Academic Press, London.