EFFECT OF MAGNESIUM CATIONS ON THE ACTIVITY AND STABILITY
1159
t, min
52°ë. The data show that the rate of inactivation
depends drastically on the pH value; an increase in the
pH to 8.0 results in a twofold increase in keff in the
presence of magnesium ions and in a tenfold increase
in the absence. In addition, the presence of magne-
sium cations extends the pH range of stability of the
enzyme.
The study performed showed that addition of mag-
nesium cations to buffer solutions or reaction mixtures
may either enhance the catalytic activity and stability of
β-galactosidases (E. coli and K. lactis) or produce no
effect on the properties of the enzyme (bovine liver and
fungous P. Òanescens β-galactosidases).
0
40
80
120
0
1
2
.8
.6
.4
4
3
2
3
.2
REFERENCES
. B. Henrissat and A. Bairoch, Biochem. J. 316, 695
1996).
. M. Ladero, A. Santos, J. L. Garcia, et al., Enzyme
Microb. Technol. 30, 392 (2002).
1
–
ln(v/v0)
1
2
3
(
Fig. 5. Kinetic curves of thermal inactivation of K. lactis
β-galactosidase (1, 2) in the absence and (3, 4) presence of
.01 mol/l magnesium cations at (2, 4) 35 and (1, 3) 40°C.
0
. P. Dutta and G. C. Majumder, Biochem. Cell Biol, 71, 22
(1993).
4
–1
4. S.-C. Li, J.-W. Han, K.-C. Chen, and C.-S. Chen, Phy-
keff × 10 , s
tochemistry 57, 349 (2001).
1
2
5
6
7
8
9
. G. S. Case and M. L. Sinnott, Biochem. J. 133, 99
1973).
. H.Y. Ryoo, E. J.Yang, H. J. Lee, et al., Biotechnol. Lett.
4, 691 (2002).
. M. N. Hung and B. N. Lee, Appl. Microbiol. Biotechnol.
8, 439 (2002).
. P. Degraeve, P. Lemay, and P. Delorme, Biochem. Bio-
phys. Acta 1292 (1), 61 (1996).
. D. B. Craig, T. Hall, and D. M. Goltz, Biometals 13, 223
(2000).
(
1
2
8
4
0
5
1
1
1
1
1
1
1
0. R. H. Jacobson, X-J. Zhang, R. F. du Bose, and
B. W. Matthews, Nature (London) 369, 761 (1994).
1. D. H. Juers, R. H. Jacobson, D. Wigley, et al., Protein
Sci. 9 (9), 1685 (2000).
2
2. C. G. Cupples, J. H. Miller, and R. E. Huber, J. Biol.
6
7
8
pH
Chem. 265 (10), 5512 (1990).
3. N. J. Roth and R. E. Huber, Biochem. Biophys. Res.
Commun. 201 (2), 866 (1994).
Fig. 6. pH dependences of effective rate constants of inacti-
vation of E. coli β-galactosidase (1) in the presence and
2) absence of magnesium cations. Phosphate buffer, 52°ë.
4. N. J. Roth and R. E. Huber, Biochem. Biophys. Res.
(
Commun. 219, 111 (1996).
5. G. S. Case and M. L. Sinnott, Biochem. J. 133, 99
(1973).
equation for various temperatures and concentrations in
the presence and absence of magnesium cations. The
inflection in the kinetic curves may point to the disso-
ciative mechanism of thermal inactivation [23]; the
effective rate constants of dissociation and denaturation
k1eff and k2eff) can be determined from the slopes of the
two segments of the experimental dependence. The
constants values are listed in Table 2.
6. T. Selwood and M. L. Sinnott, Biochem. J. 268, 317
(1990).
1
1
7. M. L. Sinnott, Chem. Rev. 90, 1171 (1990).
8. C. F. Aguilar, I. Sanderson, M. Moracci, et al., J. Mol.
Biol. 271 (5), 789 (1997).
(
1
2
2
2
2
9. M. Hidaka, S. Fushihobu, N. Ohtsu, et al., J. Mol. Biol.
3
22 (1), 79 (2002).
0. A. L. Rojas, R. A. P. Nagem, K. N. Neustroev, et al.,
J. Mol. Biol. 343, 1281 (2004).
As can be seen from Table 2 and Figs. 4 and 5, the
rate of inactivation of the enzymes decreases in the
presence of magnesium cations; the maximum effect
of magnesium cations is observed at pH > 7. Figure 6
shows the pH dependence of the effective rate con-
stants of inactivation in the presence and absence of
magnesium cations for E. coli, β-galactosidase at
1. O. M. Poltorak, E. S. Chukhrai, O. S. Pilipenko, et al.,
Zh. Fiz. Khim. (in press).
2. B. I. Kurganov, Allosteric Enzymes (Nauka, Moscow,
1
978) [in Russian].
3. O. M. Poltorak, E. S. Chukhrai, and I. Yu. Torshin,
Biokhimiya 63 (3), 360 (1998) [Biochemistry (Moscow)
63 (3), 303 (1998)].
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A Vol. 81 No. 7 2007