Spectrophotometric Determination of the Binding Constants of Succinate and Chloride to Glutamic Oxalacetic Transaminase A Biophysical Chemistry Experiment Antonio Parody-Morreale, Ana Camara-Artigas, and J& M. Sanchez-Ruiz Departmento de Quimica-Fisica, Facultad de Ciencias, 18071 Granada, Spain The function and control of biological macromolecules are mediated through their interactions with different ligands. Although increasing attention is being devoted to explaining ligand binding phenomena to biochemistry students in a basic physico-chemical level (see for example refs 1 and 21, we feel laboratory experiments to illustrate the principles are still lacking. What we present here is an experiment in which the binding constants of succinate and chloride to the enzyme glutamic oxalacetic transaminase (GOT) are measured a t a physiological pH of 7.2. The enzyme is cheaply available from commercial sources, and the binding constants can be determined by following a straightforward spectrophotometric procedure. GOT is an enzyme from the amino acid metabolism (for a review see for example ref 3) which acts as a catalyst in the reaction HOOC-CH,CH(NH,)-COOH anpartate HOOCXX-CO-COOH oxalacetate
+ HOOC-(CH,)&O-COOH
=+
u-ketoglutarate
8.1
anion in the active site none chloride succinate
Flgure 1. Protonation equilibrium of pyridoxal-5'-phosphate Schiff base In GOT activeslte. Asshown, thepKvalue depends on theanion present in the active site.
+ HOOC-(CH,),CH(NH,)-COOH glutamate
The enzyme is composed of two identical subunits of 45,000 daltons and has two active sites where the catalytically essential pyridoxal-5'-phosphate is bound t o lysine 258 through a Schiffs base linkage. Several positively charged groups (mainly arginines) can also be foundat the active site, possibly providing binding sites for the carboxylate groups of the substrate. As is to be expected, dicarboxylic acids, such as succinate, act as competitive inhibitors, although s i m ~ l anions. e such as chloride. also show significant bindingto tke active site. In the exneriment described in this paper the interaction of the enzyme with succinate a t pH 7.2 is followed spectrophotometrically using pyridoxal-5'-phosphate as a probe for the presence of the anion in the active site. GOT has a pHdependent visible spectrum (maximum at 430 nm a t acid pH and 360 nm a t basic pH), which has been attributed to the 258 protonation of the pyridoxal-5'-phosphate-lysine Schiffs base (see Figure 1). The pK value for this protonation is 5.4 in the absence of anions in the active site, but shifts to 8.1 when the enzyme is saturated with succinate. Thus, a t pH 7.2, successive additions of succinate cause a progressive increase of the band a t 430 nrn (the enzyme solution turns yellow). In Figure 2 the pyridoxal-5'-phoshate s ~ e c t r ain GOT, a t pH 7.2, both in the absence of buccinakand in the presen~eof~aturatin~amountsof it, are shown. BY following the increase of absorbance at 430 nm on the addition of suicinate to the enzyme, the equilibrium between them can he characterized. This procedure is not valid, however, for chloride, as its presence in the active site shifts the pK only to 6.3 so no change in the protonation state of the Schiff base and hence in the enzyme spectra can be detected at a pH of 7.2 after succesive additions of the anion. 988
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Figure 2. Abswption spectra of 0.7 mg/mL GOT, Hepes 40 mM. pH 7.2 in the absence (spechum I) or the presence of 50 mM succinate (spectrum 2).
In order to be able to measure the equilibrium with chloride as well, its competition with succinate is analyzed, i.e., apparent equilibrium constants for succinate in the presence of different amounts of chloride are determined. The true binding constants for both anions can then be calculated from this dependency as i t is shown in the following section. Theory
Both sites of GOT act as equivalent and independent in their bindine of succinate and chloride. As both anions eomPete for the same site in the enzyme, the equilibrium can he P, acrenresented bv the iollowina "bindinp..polvnomial", . &ding to ~ i m a nomenclature n (41,or "generating function", according to Hess and Szabo (5):
-
A portion (0.750 mL) of an enzyme solution is placed in a l-cm-path-length spectrophotometric cuvette. The enzyme concentration should be at least 1mg1mL. In order to evaluate the enzyme concentration a molar absorptivity at 280 nm of 1.4 X lo5cm-' M-I for a dimer of molecular mass of 90,000 daltons (7) can be used. Successive additions of a concentrated succinate solution into the cuvette are made. For these additions to be in the microliter range, the concentration of the succinate solution should be between 0.1 and 1M. Prior to and after each addition the enzyme spectrum in the 400-500-nm range is recorded. Figure 3 shows a typical experiment carried out by students. The additions of succinate are made up t o the point a t which no further spectral change is detected, indicating that the enzyme is saturated with the ligand.
where k, and k, are the site-binding constants for succinate and chloride and [s] and [c] their respective free conceutrations. Note that, as the sitesare equivalent and independent, the binding polynomial for the enzyme is the product of the binding polynomials for each site. At constant pressure and temperature the number of moles of succinate bound per mole of enzyme, z, or the binding parameter, can then he calculated ( 4 , 5 )as:
Dividing numerator and denominator by (1 tain
+ k,[c]) we ob-
where
is an apparent constant for the binding of succinate to the enzvme a t a eiven concentration of chloride. ~ ~ u a t i oi is n formally analogous to the equation for two equivalent and independent binding sites with constant k,'. In order to determine this one, we only have to gather data for Z, at different values of the free succinate concentration and a fixed free-chloride concentration. The constant can then be calculated graphically, as for example in a double reciprocal plot (z,-' vs. Is]-') or a Scatchard plot (z,/[s] vs. z,) (6). Once the different values of k,' have been determined at different chloride concentrations the true hinding constants for both anions can be evaluated by using eq 2. This equation can be written as
so that, from the characteristic parameters of the straight line llk,' versus [c], the binding constants k, and kc can be calculated.
Figure 3. WTabsorption spectra recorded aftersuccesive additions to 0.750 mLsolution 1 mg/mL in GOT. 2 mM in CI-. 40 mM in Heper. pH 7.2 of 2,3.5.5. 10,20, and 40 pL of 0.4 M succlnate, 2 mM CIT, 40 mM Hepes, pH 7.2 solution. Bonom spectrum abteined In the absence of succlnate and top spectrum alter the last addition of the ligand.
Every experiment of this kind is made a t a fixed chloride concentration, in the range 2-40 mM, by adding the appropriate amounts of NaCl to the enzyme and to the succinate titrating solution to maintain the same chloride concentration in both of them. Results
The absorbance change a t 430 nm is proportional to the fractional saturation of the sites with succinate. this fractional saturation being equal to the binding parameter divided by the number of sites. Thus, after correcting the absorbances measured for the dilutions made by the addition of succinate, it is possible to calculate the binding parameter using the formula:
"
-=-
Experiment
The buffer used throughout the experiment, both in the enzyme solution and in the succinate titratingagent is Hepes (N-2-hydroxyethylpiperazine-W-2-ethanesulfonic acid) 40 mM, pH 7.2, as Hepes anion does not bind significantly to the active site. Cvtonlasmic porcine heart GOT was boueht from Sigma (St. Lo&, ~ 0 ) : ~ hexperiments e were carzed out with fresh enzvme solutions. nrepared after weiahina and dialysis of the'commercial material against the Hepe: buffer: it must be noted. however, that GOTsolutionscan he stored'for several days at 4 OC o; for several months frozen with no significant alterations in catalytic or spectral properties.
2
A-A, Am-A,
whereAo and A, are the absorbances in the absence of and at saturating amounts of succinate, respectively, and A is the absorbance a t any intermediate succinate concentration. Even if the concentrations to use in the experimental analysis are those of the free species in solution, the approximation of considering the free concentration equal to the total one can be made, both with succinate and with chloride, as their concentrations during the experiments are always much higher than that of sites. The analysis of the data for the binding parameter as a function of succinate concentration obtained in the experiVolume 67
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Figure 4. Double reciprocal plot for m e data obtained in the experiment in Figure 3. A value for lhe apparent binding constant of succinate, k,', of 0.31 f 0.02 mM-' was obtained from the slooe.
ment in Figure 3 is made through a double reciprocal plot, as shown in Figure4. The apparent binding ronstant k,' for the binding of succinate is calculated from the inverse of the slooe. In order todetermine the true constants for succinate and chloride. different exoeriments at different chloride concentrationsshould be made. In our case, the class was divided into -croups - of two, to whom different chloride concentrations were assigned. The results of all the groups are set out in Figure 5, each point corresponding to one group. The analysis of the whole set of data using eq 2 yielded for the true constants the values k , = 0.35 0.05 mM-' and kc = 0.10 0.03 mM-I, which agree satisfactorily with the ones
*
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*
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F +re 5. Dspendsncy of lhe spparen D nding constants lor succinats wnh cn oriae concentrations In the experiment. True oinaing constants lor succinate and chloride determined from the sb'aight line parameters are ks = 0.35 f 0.05 mM-' and kc = 0.10 0.03 mM-'.
reported in the literature (for the cytoplasmic enzyme at pH 8.2 in Tris-cacodylate buffer k, = 0.33 mM-I and k, = 0.10 mM-I, ref 8). Acknowledgment We thank the CICYT (Grant PB87-0871) for financial support. Literature Clted 1. Freifelder. D. Physied Chemistry for Student? of Biology and Chemistry Science Books Intprnationd: Boston. 1982. 2. Klofz, 1. M. Infroduetion to Biomoleeulor Energarirs: Academic: Orlando, 1986. 3. Christen. P.; Motzler, D. E., Eda. Transominosax:Wiloy New York. 1985. 4. Wyman,J..I Mol.Biol. 1965.11.631. 5. Heaa, V. L.:Szsbo,A. J. Chrm.Edue. 1979,56,289. 6. Scatchard, G.Ann. N.Y. Acad. Sci. 1949,51,660. 7 . Feliss, N.:Martinez-Csrridn. M. Biochrm. Biophys. Chem. Commun. 1970,1O, 932. 8. Chon,S.;Michuda-Kozec, C.;Martinez-Csrribn,M.J.Biol. Chem. 1971,246,3623,
