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Studies on the NAD-specific isocitrate dehydrogenase from baker's yeast
BIOCHIMICA ET BIOPHYSICA ACTA
195
SHORT COMMUNICATIONS sc 63133
Studies on the NAO-specific isocitrate dehydrogenase from baker's yeast The NAD-speciftc isocitrate dehydrogenase (Ls-isocitrate:NAD oxidoreductase (decarboxylating), EC 1.1.1.41) from diverse biological sources l - 3 does not obey the classical Michaelis-Menten kinetics, since plots of activity against isocitrate concentration yield sigmoid curves. With the yeast enzyme", as well as with the enzyme from Neurospora crassa», AMP or citrate show an activating effect at low substrate concentrations. It has been suggested-." that these effects may have a regulatory role in the tricarboxylic acid cycle, and that the behavior of the NAD-specific isocitrate dehydrogenase may be explained, according to the mechanism proposed for many regulatory enzymes-, by assuming the existence on the enzymic protein of an "allosteric site", able to bind the various "effectors" (including the substrate itself), and consequently to enhance the affinity of the active site for the substrate, by a modification of the protein conformation. We wish to present a preliminary report of results obtained with the NADspecific isocitrate dehydrogenase from baker's yeast. The enzyme was purified from baker's yeast according to KORNBERG AND PRICER5 ; all the experiments were carried out with the "Cy eluate", which corresponded to a purification of about 40-fold. The protein concentration was about 0.8 mgjrnl, The enzymic activity was determined by measuring the rate of NAD reduction at 366 mt-' with an Eppendorf Photometer, in cuvettes of ro-mm light path, at room temperature. The final volume of the reaction mixtures was 3 ml, the pH was 7.6, and 0.33 mM NAD was present in every case. DL-Isocitric acid trisodium salt was used as substrate, but the concentrations given are those of a single isomer. The detailed composition of the reaction mixtures is reported in the individual experiments. The reaction was started with 0.04 ml of "Cy eluate" and the increase in absorbance at 366 mt-' was followed by readings at I5-sec intervals. The results are expressed in activity units: one unit is defined as an increase in absorbance of o.oor/min. The NAD-specific isocitrate dehydrogenase from yeast requires a divalent metal ion: Mg2+, Mn 2+ , Zn 2+ and C0 2+ have been found effective to different degrees'.", We have observed that the relative activities of these ions depended on the substrate concentration; if the substrate concentration was lower, M 2+ was found to be the less effective activator. At 0.33 mM isocitrate, with O.I M Tris buffer (for the influence of the buffer concentration, see below), no activity was observed with 3.3 mM Mg2+ in the absence of AMP (Table I). However, a cooperative effect, resulting in considerable enzymic activity, was obtained when 0.083 mM Zn2+ was added together with 3.3 mM Mg2+. On the other hand, the presence of Mg2+ together with 'Mn2+ yielded a strong inhibition of the activity observed with Mn2+ alone. These effects were not evident at 3 mM isocitrate concentration. As reported in Table II, we have found that the isocitrate dehydrogenase was strongly inhibited by NaCl. This inhibition was dependent on the concentration of the metal ion used as activator, and Mg2+ was found to be the less effective in this respect. Furthermore, rn the presence of 3.3 mM Mg2+, the addition of AMP or of 0.17 mM Zn 2+ removed the inhibition observed when only Mg2+ or Zn 2+ was present.
g
Biochim, Biophys, Acta, lIO(1965) 195-197
196
SHORT COMMUNICATIONS
TABLE I EFFECTS OF METAL ACTIVATORS ON ISOCITRATE DEHYDROGENASE
The reaction mixtures contained, in a final volume of 3 ml: 0.1 M Tris-HC] buffer (pH 76), 0.33 roM NAD, 0.04 rnl of "C,.. eluate", isocitrate and other additions as indicated.
Isocitraie concentration (mM)
Activity in the pre.set/ce of 0.67 m M MnZ+ 0.083 mM ZnZ+ 3.3 mll/f Mg2+ alone
with
with
0.I7 mM 0.083 mM
Zn H
AMP 66
3 0·33
23 8
41
42
61
43 43
1
with o.67mM Mn H
II
Thus, in this case 'too, a cooperative effect was obtained with Mg2+ and Zn 2 +, but not with Mg 2+ and Mn 2+ . The inhibitory effect of NaCI, also observed with other salts, appeared to be non-specifically dependent on the salt concentration, since a similar inhibition could be obtained with high concentrations of the Tris buffer. The dependence of the enzymic activity on the buffer concentration is shown in Fig. I; the effect of the buffer concentration was enhanced by lowering the concentration of isocitrate and was abolished by AMP. Similar results were obtained with phosphate buffer. We found that HgCl 2 also inhibited the enzyme (Table II): AMP and Zn2+ had effects similar to those observed in the case of inhibition by NaCl. TABLE II EFFECTS OF METAL IONS ON THE INHIBITION OF ISOCITRATE DEHYDROGENASE BY
NaCl
AND
HgCl 2
The reaction mixtures contained, in a final volume of 3 ml: 0.1 M Tris-H'Cl buffer (pH 7.6), 0.33 mM NAD, 0.04 ml of "C,.. eluate" and 3 mM isocitra'te, Other additions as indicated.
Additions
Activity without inhibitor
3.3 mM 0.17 mM 0.67 mM 0.17 mM 0.67mM 3.3 mM 3.3 mM 3.3 mM
Mg2+ Zn2+ Zno+ MnH Mn H MgH Mg2+ MgH
+ 0.17 mM AMP + 0.17 mM Zn H
+ 0.17 mM Mn2+
42 34 39 63 66 43 44
52
with o.I3 M
with I.? f.tM
NaCl
HgCl 2
3 7 26 7 39 41 36 10
6 7 33 12 51 35 37 14
The complex behavior of NAD-specific isocitrate dehydrogenase from yeast, as evident from previous research- and from the present experiments, could possibly be explained on the basis of the maintenance of, or alteration of, a particular conformation of the enzymic protein, firstly perhaps with respect to the binding of the metal Biochim, Biophys. Acta,
IIO
(1965) 195-197
197
SHORT COMMUNICATIONS
'"
i...
"
:>
,:
... >
t-
30
V C
. I.
TR1S BUFFER.ml
Fig. I. Effects of the concentration of the Tris buffer on the activity of isocitrate dehydrogenase. The reaction mixtures contained, in a final volume of 3 ml: 0.33 mM N AD, 3.3 mM MgH, 0.04- ml "C y eluate" and the indicated volumes of 0.2 M Tris-I-ICI buffer (pH 7.6). Isocitrate concentrations: 3 mM, e; I mM,.; I mM (with 0.17 mM AMP), 0; 0.33 mM, ....
ion activator. However, the features of such a mechanism can only be a matter for speculation at present. Our results indicate that the buffers seem to have an inhibitory effect on the activity of the enzyme. Therefore, it appears likely that in vitro it is only possible to study the kinetic behavior of the inhibited enzyme. In that case, the deviation from the Michaelis-Menten kinetics could also be explained on kinetic grounds? without assuming the existence of another site, i.e. the "allosteric site", capable of binding the substrate. Institute of Biological Chemistry and Institute of Human Physiology, University of Modena, Modena (Italy) I
J.
C. CENNAMO
G. G.
MONTECUCCOLI BONARETTI
.f.
A. HATHAWAY AND D. E. ATKINSON, Biol, Chern., 23 8 (1963) 2875SANWAL AND C. S. STACHOW, Biochim, Biophys. Acta, 96 (1965) 28. H. GOEBELL AND M. KLINGENBERG, Biochem. Z., 340 (1964) 44 1 . J. MONOD, ]. P. CHANGEUX AND F. JACOB, Mol. Biol., 6 (19~3) 306. A. KORNBERG AND W. E. PRICER, Bioi. Chem., 189 (195 1 ) 123 . C. FRIEDEN, Biol, Chem., 239 (1964) 35 2 2 •
2 B. D.
3 4 5 6
J.
J.
J.
Received July 5th, 1965 Biochim: Biophys. Acta,
110
(19 65) 195-197
195
SHORT COMMUNICATIONS sc 63133
Studies on the NAO-specific isocitrate dehydrogenase from baker's yeast The NAD-speciftc isocitrate dehydrogenase (Ls-isocitrate:NAD oxidoreductase (decarboxylating), EC 1.1.1.41) from diverse biological sources l - 3 does not obey the classical Michaelis-Menten kinetics, since plots of activity against isocitrate concentration yield sigmoid curves. With the yeast enzyme", as well as with the enzyme from Neurospora crassa», AMP or citrate show an activating effect at low substrate concentrations. It has been suggested-." that these effects may have a regulatory role in the tricarboxylic acid cycle, and that the behavior of the NAD-specific isocitrate dehydrogenase may be explained, according to the mechanism proposed for many regulatory enzymes-, by assuming the existence on the enzymic protein of an "allosteric site", able to bind the various "effectors" (including the substrate itself), and consequently to enhance the affinity of the active site for the substrate, by a modification of the protein conformation. We wish to present a preliminary report of results obtained with the NADspecific isocitrate dehydrogenase from baker's yeast. The enzyme was purified from baker's yeast according to KORNBERG AND PRICER5 ; all the experiments were carried out with the "Cy eluate", which corresponded to a purification of about 40-fold. The protein concentration was about 0.8 mgjrnl, The enzymic activity was determined by measuring the rate of NAD reduction at 366 mt-' with an Eppendorf Photometer, in cuvettes of ro-mm light path, at room temperature. The final volume of the reaction mixtures was 3 ml, the pH was 7.6, and 0.33 mM NAD was present in every case. DL-Isocitric acid trisodium salt was used as substrate, but the concentrations given are those of a single isomer. The detailed composition of the reaction mixtures is reported in the individual experiments. The reaction was started with 0.04 ml of "Cy eluate" and the increase in absorbance at 366 mt-' was followed by readings at I5-sec intervals. The results are expressed in activity units: one unit is defined as an increase in absorbance of o.oor/min. The NAD-specific isocitrate dehydrogenase from yeast requires a divalent metal ion: Mg2+, Mn 2+ , Zn 2+ and C0 2+ have been found effective to different degrees'.", We have observed that the relative activities of these ions depended on the substrate concentration; if the substrate concentration was lower, M 2+ was found to be the less effective activator. At 0.33 mM isocitrate, with O.I M Tris buffer (for the influence of the buffer concentration, see below), no activity was observed with 3.3 mM Mg2+ in the absence of AMP (Table I). However, a cooperative effect, resulting in considerable enzymic activity, was obtained when 0.083 mM Zn2+ was added together with 3.3 mM Mg2+. On the other hand, the presence of Mg2+ together with 'Mn2+ yielded a strong inhibition of the activity observed with Mn2+ alone. These effects were not evident at 3 mM isocitrate concentration. As reported in Table II, we have found that the isocitrate dehydrogenase was strongly inhibited by NaCl. This inhibition was dependent on the concentration of the metal ion used as activator, and Mg2+ was found to be the less effective in this respect. Furthermore, rn the presence of 3.3 mM Mg2+, the addition of AMP or of 0.17 mM Zn 2+ removed the inhibition observed when only Mg2+ or Zn 2+ was present.
g
Biochim, Biophys, Acta, lIO(1965) 195-197
196
SHORT COMMUNICATIONS
TABLE I EFFECTS OF METAL ACTIVATORS ON ISOCITRATE DEHYDROGENASE
The reaction mixtures contained, in a final volume of 3 ml: 0.1 M Tris-HC] buffer (pH 76), 0.33 roM NAD, 0.04 rnl of "C,.. eluate", isocitrate and other additions as indicated.
Isocitraie concentration (mM)
Activity in the pre.set/ce of 0.67 m M MnZ+ 0.083 mM ZnZ+ 3.3 mll/f Mg2+ alone
with
with
0.I7 mM 0.083 mM
Zn H
AMP 66
3 0·33
23 8
41
42
61
43 43
1
with o.67mM Mn H
II
Thus, in this case 'too, a cooperative effect was obtained with Mg2+ and Zn 2 +, but not with Mg 2+ and Mn 2+ . The inhibitory effect of NaCI, also observed with other salts, appeared to be non-specifically dependent on the salt concentration, since a similar inhibition could be obtained with high concentrations of the Tris buffer. The dependence of the enzymic activity on the buffer concentration is shown in Fig. I; the effect of the buffer concentration was enhanced by lowering the concentration of isocitrate and was abolished by AMP. Similar results were obtained with phosphate buffer. We found that HgCl 2 also inhibited the enzyme (Table II): AMP and Zn2+ had effects similar to those observed in the case of inhibition by NaCl. TABLE II EFFECTS OF METAL IONS ON THE INHIBITION OF ISOCITRATE DEHYDROGENASE BY
NaCl
AND
HgCl 2
The reaction mixtures contained, in a final volume of 3 ml: 0.1 M Tris-H'Cl buffer (pH 7.6), 0.33 mM NAD, 0.04 ml of "C,.. eluate" and 3 mM isocitra'te, Other additions as indicated.
Additions
Activity without inhibitor
3.3 mM 0.17 mM 0.67 mM 0.17 mM 0.67mM 3.3 mM 3.3 mM 3.3 mM
Mg2+ Zn2+ Zno+ MnH Mn H MgH Mg2+ MgH
+ 0.17 mM AMP + 0.17 mM Zn H
+ 0.17 mM Mn2+
42 34 39 63 66 43 44
52
with o.I3 M
with I.? f.tM
NaCl
HgCl 2
3 7 26 7 39 41 36 10
6 7 33 12 51 35 37 14
The complex behavior of NAD-specific isocitrate dehydrogenase from yeast, as evident from previous research- and from the present experiments, could possibly be explained on the basis of the maintenance of, or alteration of, a particular conformation of the enzymic protein, firstly perhaps with respect to the binding of the metal Biochim, Biophys. Acta,
IIO
(1965) 195-197
197
SHORT COMMUNICATIONS
'"
i...
"
:>
,:
... >
t-
30
V C
. I.
TR1S BUFFER.ml
Fig. I. Effects of the concentration of the Tris buffer on the activity of isocitrate dehydrogenase. The reaction mixtures contained, in a final volume of 3 ml: 0.33 mM N AD, 3.3 mM MgH, 0.04- ml "C y eluate" and the indicated volumes of 0.2 M Tris-I-ICI buffer (pH 7.6). Isocitrate concentrations: 3 mM, e; I mM,.; I mM (with 0.17 mM AMP), 0; 0.33 mM, ....
ion activator. However, the features of such a mechanism can only be a matter for speculation at present. Our results indicate that the buffers seem to have an inhibitory effect on the activity of the enzyme. Therefore, it appears likely that in vitro it is only possible to study the kinetic behavior of the inhibited enzyme. In that case, the deviation from the Michaelis-Menten kinetics could also be explained on kinetic grounds? without assuming the existence of another site, i.e. the "allosteric site", capable of binding the substrate. Institute of Biological Chemistry and Institute of Human Physiology, University of Modena, Modena (Italy) I
J.
C. CENNAMO
G. G.
MONTECUCCOLI BONARETTI
.f.
A. HATHAWAY AND D. E. ATKINSON, Biol, Chern., 23 8 (1963) 2875SANWAL AND C. S. STACHOW, Biochim, Biophys. Acta, 96 (1965) 28. H. GOEBELL AND M. KLINGENBERG, Biochem. Z., 340 (1964) 44 1 . J. MONOD, ]. P. CHANGEUX AND F. JACOB, Mol. Biol., 6 (19~3) 306. A. KORNBERG AND W. E. PRICER, Bioi. Chem., 189 (195 1 ) 123 . C. FRIEDEN, Biol, Chem., 239 (1964) 35 2 2 •
2 B. D.
3 4 5 6
J.
J.
J.
Received July 5th, 1965 Biochim: Biophys. Acta,
110
(19 65) 195-197