RESEARCH
Reconstituted Enzyme Has Original Activity Dissociation and reconstitution of aldolase lead to better understanding of the enzyme's molecular structure Aldolase, the enzyme that catalyzes the reaction of fructose-1,6-diphosphate to D-glyceraldehyde phosphate plus dihydroxyacetone phosphate, has been split into three polypeptide subunits by acid, urea, or sodium dodecyl sulfate'(C&EN, Nov. 26, page 4 7 ) . And the subunits can be reassociated under relatively mild conditions to form a macromolecule that is almost identical in physical characteristics and enzymic activity to the original enzyme, say Dr. E. Stellwagen and Dr. H. K. Schachman of the University of California's biochemistry and virus laboratory, Berkeley. The three polypeptide subunits appear to be in the form of random chains. In aldolase, they are held together by noncovalent forces and folded into compact, relatively rigid particles. Until recently, attempts to reconvert denatured proteins into macromolecules having physical, chemical, and biochemical properties similar to the original material have been unsuc-
ENZYME STUDY. Dr. E. Stellwagen (left) and Dr. H. K. Schachman prepare ultracentrifuge for studies of dissociation and reconstitution of aldolase 40
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cessful. Biochemists generally believed that denaturation is an irreversible process because of the seeming improbability of the specific refolding of the randomly coiled polypeptide chains. Recently, ribonuclease studies by Dr. F. H.'White, Jr., Dr. C. B. Anfinsen, and co-workers at the National Institutes of Health, Bethesda, Md. [/. Biol Chem., 234, 1353 (1961); Troc. Natl Acad. Sci. U.S., 47, 1309 (1961)], show that an enzyme consisting of a single polypeptide chain with disulfide cross-linking could be disorganized and denatured by rupture of these bonds in the presence of urea. The original enzymic activity can be restored by reforming the disulfide groups through oxidation.
Illinois independently obtained similar results [Fed. Troc, 2 1 , 254 (1962)]. Other studies on multichain en zymes have been made recently by Dr. C. Levinthal, Dr. E. R. Signer, and Dr. K. Fetherolf at Massachusetts Institute of Technology. They made similar reconstitution experiments on alkaline phosphatase [Troc. Natl Fed. Sci., 48, 1230 (1962)]. And Dr. D. Young and co-workers at NIH have studied myosin [/. Biol. Chem., 237, 3116 (1962)]. These workers, how ever, couldn't recover the original bio logical activity of myosin under their experimental conditions. Support. Considerable evidence supports the belief that the reactivated material is the same as the original enzyme :
These results are significant because random cross-linking between the sulfur atoms with only a 1% recovery of enzyme activity occurs if urea is present during oxidation. This indicates that during the normal oxidative reconstitution, a directing influence and definite cross-linking pattern are important to regain the molecular constitution and activity of the original enzyme. The experiments of Dr. T. Isemura and co-workers at Osaka University, Osaka, Japan, on lysozyme and taka-amylase coincide with these results [Biocheîn. Biophys. Res. Commun., 5, 373 (1961)].
• The sedimentation rate and ultracentrifuge pattern for the reconsti tuted protein are the same as for aldo lase. • The molecular weights of the two macromolecules are almost identical. • Optical rotatory dispersion and spectral patterns of the reconstituted protein and of aldolase are identical.
In view of the success encountered in regenerating active enzymes composed of a single polypeptide chain, Dr. Stellwagen and Dr. Schachman decided to study a more complex enzyme. Working with aldolase isolated from rabbit muscle, they split the enzyme (without rupturing any covalent bonds) into three polypeptide subunits devoid*of enzymic activity [Biochemistry, 1, 1056 (1962)]. And they can regroup the polypeptide chains to give & macromolecule having aldolase activity by removing the inactivating agent, using either dilution or dialysis. Dr. W. C. Deal and Dr. Κ. Ε. Van Holde at the University of
Addition of hydrochloric acid, ace tic acid, urea, or sodium dodecyl sul fate to aldolase in solution ruptures the protein molecule and destroys its enzymic properties. Dr. Stellwagen and Dr. Schachman routinely use two types of kinetic assays to determine aldolase activity. Both involve the spectrophotometry detection of triose phosphates formed by cleavage of fructose-l,6-diphosphate. Physical measurements include sedimentation rates, viscosity, and optical rotatory dispersion determinations. An acid medium (pH 2.0) or 4M urea at p H 5 converts aldolase into units of molecular weight 0.46 X 10 5 ,
•Finally, it has the same specific activity as native aldolase; the 16 sulfhydryl groups normally masked in al dolase and made available only on denaturation are again masked in the reconstituted macromolecule.
Sedimentation Velocity of Aldolase Changes with Urea Concentration
Sedimentation velocity of aldolase changes with urea concentration, as shown by these patterns. The sedimentation pattern in 1 represents native aldolase with no urea present (sedimentation coefficient is 7.6 S). Reconstituted enzyme (5) gives an identical pattern. With 1.5M urea (2), two peaks show up: 2.1 S and 6.4 S. Area of the two boundaries shows that about one third of the enzyme is at the subunit level; why there's a 6.4 S value instead of 7.6 S
about a third of the molecular weight value of the original aldolase. The Berkeley scientists attribute the acid dissociation to the large intramolecular repulsive forces that develop as the net charge is increased by titration of the carboxylate ions. At an ionic strength of 0.05, the transition from the native to the dissociated structure occurs with a midpoint about pH 4. This pH value corresponds to the pK normally assigned to carboxyl groups in proteins, they note. As for aldolase's sensitivity to urea, 4M urea causes it to dissociate. A 2M concentration isn't enough to cause complete dissociation; the partially disordered protein molecules aggregate to form particles larger than the native enzyme. This is probably due to the availability of interacting groups when the polypeptide chains unfold. In the absence of sufficient urea, these cause aggregation. At higher urea concentrations ( 4 M ) , interchain interactions are reduced and the single polypeptide chains represent the stable conformation. The exact mechanism of the urea dissociation isn't clear, even though urea has been used as a dénaturant for many years, Dr. Stellwagen and Dr. Schachman point out. The disruptive effect might be the result of the breaking of intra- and interchain hydrophobic or hydrogen bonds. Dilute acetic acid (0.83M, p H 2.6) causes aldolase dissociation, probably by rupturing hydrophobic bonds in macromolecules, as well as by gener-
for unchanged enzyme is still under study. In 3, 1.6 S represents the sedimentation velocity of the polypeptide subunit formed in 2.0M urea; 11.0 S is the sedimentation velocity of the aggregated material when urea concentration isn't enough to cause complete dissociation. With 4.0M urea, complete dissociation takes place with a sedimentation value of 1.5 S (4). Reconverting the subunits by dialysis gives a macromolecule having the same sedimentation value
ating large intramolecular repulsive forces at low pH. Sodium dodecyl sulfate's effective denaturing property is partly attributed to the repulsive forces produced when it binds to the macromolecule, and to the rupture of hydrophobic bonds by interaction with the detergent's long hydrocarbon chain. The denatured enzyme's sedimentation velocity pattern is different from aldolase's. With increased acidity or urea concentration, there's an accompanying disappearance of material from the boundary corresponding to native aldolase. At the same time, a material with a slower sedimentation rate (indicating lower molecular weight) appears. Aldolase in the ultracentrifuge has a single symmetrical boundary corresponding to a sedimentation coefficient of 7.6 S when measured at 59,780 r.p.m. (25° C ) . At pH 2.0 or in the presence of 4M urea, this is replaced by a boundaiy having a sedimentation rate of 1.5 S. Dr. Stellwagen and Dr. Schachman point out that this decreased sedimentation coefficient can be caused by a dissociation of macromolecules into unfolded subunits. Viscosity studies support this idea. The value for the reduced viscosity increases from 4.0 ml. per gram for native aldolase (pH 7) to about 20 ml. per gram when acid or urea is added, indicating a marked swelling or elongation of the molecules. Optical rotatory dispersion shows that [α]Ό changes from —23° for
native aldolase to - 6 2 ° at pH 2.0 and to —83° in 4M urea. Similarly, the Drude constant (λ6.) changes from 283 χημ. to 238 χημ. at -pH 2.0 and to 223 m/A. in 4M urea. According to current views, these results show that the polypeptide chains have undergone a marked conformational change from an organized structure (in native al dolase) to one of greater randomness. While there is still doubt about the exact shape and effective volume of the dissociated polypeptide chains, their hydrodynamic properties indicate they are disorganized, flexible, coil like chains. The large dependence of sedimentation coefficient on concen tration characterizes random chain polymers in good solvents. Also, the maximum in the concentration de pendence of the subunits' reduced vis cosity in acid is similar to that for polyelectrolytes at low concentration. And it indicates the deform ability of the randomly-coiled chains, depending on local ionic environment, Dr. Stell wagen and Dr. Schachman say. Whether the three polypeptide subunits are identical isn't clear. Frac tionation experiments, amino acid anal yses, and spectrophotometric titration of the tyrosyl residues aren't definite. In molecular weight determinations (from sedimentation experiments on dissociated aldolase), the plot of the log of protein concentration against the square of the distance from the rota tion axis shows a slight curve, not the straight line that would be expected if the chains were of the same molecular DEC.
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ALLIS42
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CHALMERS
weight. However, Dr. Stellwagen and Dr. Schachman suspect this could be caused by slight aggregation of the chains, and isn't a reflection of chain length difference among the dissociated subunits. The two Berkeley scientists point to the findings of Dr. A. Kowalsky and Dr. P. D. Boyer. Working at the University of Minnesota on the digestion of rabbit muscle aldolase with carboxypeptidase in H 2 0 1 8 , the Minnesota workers identified at least three carboxyl-terminal tyrosine residues []. Biol Chem., 235, 604 ( I 9 6 0 ) ] . This would be consistent with the view that the three chains are identical. However, they also identified two alanine residues in the interior of the chain (or chains) just before one or two of the tyrosine residues. This suggests that alanine is a penultimate residue at the carboxyl-terminal end in at least one of the chains, and that all three chains may not be identical. Enzyme Reconstitution. Rapid dilution of the dissociated aldolase causes a 65% recovery of the original aldolase activity. This is best accomplished by pouring the p H 2 solution into a large excess of buffer solution (pH 5.5). With augea-dissociated aldolase, the enzymic activity is recovered through rapid dilution or dialysis. This agrees with the findings of Dr. A. D. Swenson and Dr. Boyer at Minnesota, who found that solutions of aldolase in 4M urea regain activity on dilution to 0.08M urea at p H 7.2 [JACS, 79, 2174 (1957)]. "It seems that for aldolase, as for other enzymes, the native structure has the conformation of lowest free energy, and that the type and sequential arrangement of the amino acids in the polypeptide chains dictate the refolding and reassociation of the subunits," Dr. Stellwagen and Dr. Schachman note. The rapidity of the restoration of enzymic activity indicates that extensive annealing isn't necessary. Preliminary analyses of the kinetics of the reassociation point to a first-order reaction with respect to concentration. A relatively slow conformational change within each subunit, followed by a more rapid association process, could account for these findings, Dr. Stellwagen and Dr. Schachman say. Or the final annealing of the structure after association of the three chains may be rate determining, they add. They are now studying the reconstitution process.
