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Hughes, T. R., and Klotz, I. M. (1956), Methods Biochem. Anal. 3,265. Joyce, B. K., andCohn, M. (1969), J. Biol. Chem. 244,811. Mildvan, A.S.,and Cohn, M. (1963), Biochemistry 2,910. Shulman, R. G., Sterlicht, H., and Wylunda, B. J. (1965),
J. Chem. Phys. 43,3116. Takahashi, T., Irie, M., and Ukita, T. (1967), J . Biochem. (Tokyo)61,669. Ukita, T., Takahashi, T., Waku, J., and Hoshino, 0. (1964), J. Biochem. (Tokyo)55,293.
Spin-Labeling Studies of Aspartate Transcarbamylase. I. Effects of Nucleotide Binding and Subunit Separation* Trent Buckman'
ABSTRACT : Aspartate transcarbamylase has been reacted with the spin-labeling reagent 4-a-bromoacetamido-2,2,6,6tetramethylpiperidine-N-oxy1 and the effects of ligand binding, pH, and separation into subunits on the electron spin resonance spectrum of the labeled protein have been investigated. CTP and ATP binding cause broadening of the electron spin resonance spectrum while UTP and succinate (a substrate analog) and carbamyl phosphate have no effect.
T
he spin-labeling technique, in which conformation changes in biopolymers are followed by means of changes in the electron spin resonance spectrum of a bound freeradical label, has been shown by McConnell and coworkers to be very useful in studying the allosteric transitions accompanying oxygen binding in hemoglobin (Ogawa and McConnell, 1967; McConnell and Hamilton, 1968; McConnell et af., 1969). In these investigations the protein was reacted with the reagent N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide (I) and a change in the electron spin resonance spectrum of the label bound at the 093 cysteine was observed which correlated with the degree of oxygenation in agreement with the predictions of the sequential model for allosteric transitions of Koshland et af. (1966) (referred to as K N F in this paper). NH COCHzI
I
0
I
The aim of the present work is to use this technique to study the binding of allosteric effectors in a more complex system which exhibits heterotropic as well as homotropic
* From
~~
the Biochemistry Division, Department of Chemistry and Chemical Engineering, University of Illinois, Urbana, Illinois 61801, Received January 21, 1970. This research was supported by United States Public Health Service Grant USPH RG 7768. United States Public Health Service Special Fellow, 1969.
Increasing pH and decomposition into subunits byp-hydroxymercuribenzoate result in narrowing of the spectrum. It was also found that the course of the labeling reaction was affected by the presence of ligands. The conformational change measured by changes in the labeled protein electron spin resonance in the presence of CTP did not correlate well with the predictions of the simple allosteric models for heterotropic effects.
effects. An ideal protein for such a study is the regulatory enzyme aspartate transcarbamylase (ATCase) from Escherichia coli which exhibits all of the general features of allosterism, including cooperativity in substrate binding, and inhibition and activation through binding at sites distinct from the catalytic site. With the exception of hemoglobin it is the most extensively studied protein in regard to its allosteric properties and is available in gram quantities from a special strain of Escherichia coli by a relatively simple isolation procedure (Gerhart and Holoubek, 1967). ATCase is the first enzyme concerned only with pyrimidine biosynthesis and is subject to feedback inhibition by the end product cytidine triphosphate (CTP), as a means of turning off synthesis of intermediates when levels of the end product are sufficiently high in the cell. It is activated in a manner analogous to the CTP inhibition by adenosine triphosphate (ATP). This has been suggested to be of importance in maintaining the balance between the supply of purines and pyrimidines in the nucleic acid synthesis pool (Gerhart and Pardee, 1962). Also binding of the substrates aspartate and carbamyl phosphate (CAP)* is cooperative through interactions between catalytic sites on different subunits. Considerable progress has been made toward understanding the structure and mechanism of catalysis by ATCase in recent years. Gerhart and Schachman (1965) have shown that the enzyme can be separated by heat or mercurials into 2 distinct types of subunits, one of which binds only the substrates and is fully catalytically active (catalytic C) and another that has no activity and binds the inhibitor Abbreviations used are: CAP, carbamyl phosphate, p-hydroxymercuribenzoate.
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CTP (regulatory R). They determined molecular weights and ligand binding constants for the subunits and o n the basis of these proposed a structural model for the enzyme consisting of 2 C subunits with 2 catalytic sites each and 4 R subunits with 1 regulatory site each. The R subunit has recently been sequenced (Weber, 1968b) and preliminary X-ray studies o n the intact enzyme have been reported (Wiley and Lipscomb, 1968). The results of these studies suggest that the enzyme has a threefold axis of symmetry and is composed of 6 identical regulatory chains and 6 identical catalytic chains, in conflict with the earlier findings. Recently two research groups have reported detailed investigations of the conformation changes that accompany binding of the allosteric ligands. One approach used by Gerhart and Schachman (1968) involved the observation of changes that occur in the sedimentation velocity of the protein, and its reaction rate with p-hydroxymercuribenzoate, the reagent used to separate the C and R subunits, as a result of changes in the accessibility of potential sites of attack in different conformational states. The sedimentation coefficient decreases by about 3.6% in the presence of the substrate CAP and succinate, a n inactive analog of the other substrate aspartate. This change can be partially reversed by CTP, but CTP alone has no effect. Similarly, the rate of reaction with PMB increases in the presence of CAP and succinate and this increase can be partially reversed by CTP although CTP itself does not affect the rate. McClintock and Markus (1968) used changes in the rate of reaction of ATCase with proteolytic enzymes in the presence of the allosteric ligands as a measure of conformational state. I n these studies it was found that the rate of digestion by several such enzymes was increased by the substrate aspartate and decreased by both the inhibitor CTP and the activator ATP. All of these results, with the exception of the effect of ATP o n digestability, are consistant with allosteric activation resulting in a more open (relaxed) protein structure and inhibition in a more constrained structure. From a comparison of ligand binding curves with the state functions for the changes in sedimentation coefficient and reaction rate with PMB in the presence of CAP, succinate, and CTP, Changeux and Rubin were able to calculate a set of parameter values to fit the behavior of ATCase to the Monod ef ul. (1965) (MWC) concerted allosteric model. However, McClintock and Markus (1969) found that the state function determined from the effect of the substrate aspartate on digestability was homologous with plots of activity and binding as a function of aspartate concentration and thus concluded that in regard to the homotropic effects the behavior of ATCase was more consistant with the KNF sequential model. The spin-labeling technique offers a number of unique advantages as a probe to investigate the details of these allosteric conformation changes. The observed changes in the electron spin resonance spectrum can be unambiguously related to local conformational changes in the region of the protein where the label is attached and, to the extent that the location of the label can be determined, the portion of the protein undergoing the conformation change can also be specified. In most cases where this technique has been used it has been found that the labeling agent does not seriously alter the specific activity of the enzyme and thus perturbation of the label spectrum can easily be followed under
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the normal conditions of catalysis. From the nature of the change in the electron spin resonance spectrum it may be possible to reach conclusions as to how the protein tertiary structure is modified by ligand binding. In this paper the results of electron spin resonance studies on intact ATCase labeled with the reagent, 4-a-bromoacetamido-2,2,6,6-tetramethylpiperidine-N-oxyl(II) are presented. NHCOCH2Br
A
I
>LN,< 0
I1
This labeled protein shows no loss of activity o r susceptability to CTP and the nitroxide electron spin resonance spectrum is sensitive to heterotropic allosteric effects. The effects of CTP, ATP, UTP, and substrate binding, p H changes, and reaction with PMB have been investigated. It has also been found that the type of electron spin resonance spectrum exhibited by the labeled ATCase can be modified by carrying out the labeling reaction in the presence of various ligands. These results are discussed in terms of their relation to the other studies of conformational effects in ATCase and the MWC and K N F allosteric models. Future papers in this series will be concerned with the results of investigations with other labeling agents and labeling of individual subunits, in addition to further explorations with this labeled enzyme. Materials and Methods Chemicals. PMB and ATP were purchased from Sigma Chemical Co., UTP from California Biochemical Research, CTP from Pierce Chemical Co.. and N-ethylmaleimide from Aldrich Chemical Co. The spin-labeling reagent I1 was prepared by a method similar to that used by Ogawa and McConnell (1967), and which has been described previously by Buckman et d. (1969). Enzyme. ATCase was isolated from a special strain of E. coli following the procedure of Gerhart and Holoubek (1967). Bacteria were grown at 35" in 100 1. of media to it density of about 200 klett unitdm1 and disrupted by sonic oscillation. The strain of E. coli was kindly provided by Dr. John Gerhart. The purified enzyme was stored frozen at -15' in pH 7.0, 0.04 M potassium phosphate buffer containing 2 X 2-mercaptoethanol and 2 X 10F4 M sodium EDTA. Protein concentration in crude extracts was determined using the method of Lowry et a / . (1951) with bovine serum albumin as a reference standard. The concentration of purified ATCase was determined from measurement of the optical density at 280 nm using value of 0.59 for the absorbance of I-mg /ml solution (Gerhart and Schachman, 1968). Activity of the enzyme was measured using the colorimetric assay for carbamyl aspartate of Gerhart and Pardee (1962). A maximum specific activity of 7900 units,'mg was obtained for the purified preparation. In the presence of hi CAP the ATCase M aspartate and 3.6 >( 5 X
SPIN-LABELING STUDIES OF
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of bound nitroxide can then be estimated by comparison was inhibited 54% by 5 x M CTP under the usual assay with such a solution of known concentration. conditions. Separation of the Subunits. The C and R subunits of native Enzyme purity was also checked by gel electrophoresis and spin-labeled ATCase were separated by reaction with and the ultracentrifuge sedimentation velocity pattern. excess PMB and isolated o n a column of DEAE-Sephadex Acrylamide gels (7.5%) run in pH 7.0 barbitol buffer at A-50 using the procedures described by Gerhart and Holou25-30 V/cm showed, in addition to the ATCase band, a bek (1967). In the experiment in which the isolated subunits small slower moving band (1 to 5 %) and occasionally traces from spin-labeled protein were examined for electron spin of a component running just ahead of ATCase. Relative resonance signals the 2-mercaptoethanol stabilizer was not concentrations were measured with a densitometer. ATCase used to prevent possible nitroxide reduction. The R subunit was also denatured to the C and R peptide chains with sodium had the correct sedimentation coefficient in the subunit dodecyl sulfate and the denatured protein run on sodium mixture and appeared in the correct place on the Sephadex dodecyl sulfate containing acrylamide gels. The molecular column, but appeared to have aggregated when examined weights of the chains from their movement on the gels in the analytical ultracentrifuge and by gel electrophoresis relative to reference standards agree with those found by after isolation. Weber (1968b) using this method. CTP Binding Study. Binding of CTP was investigated In the analytical ultracentrifuge with schlieren optics using a series of solutions containing 1.5 mg/ml of ATCase the ATCase preparation had a major component with a and concentrations of CTP from 4.5 X 10-6 M to 2.1 X 10-4 sedimentation coefficient szo,w = 11.2 S in good agreement M. The CTP concentration in these solutions was deterwith the value of 11.7 S for ATCase determined by Gerhart mined from the absorbance at 271 nm before addition of the and Schachman (1965) and a minor component (
