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BIOCATALYSIS-2000: FUNDAMENTALS & APPLICATIONS

127

STABILIZATION MECHANISM OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE
E. A. Zaitseva, E. S. Chukhrai, and O. M. Poltorak

Yeast glucose-6-phosphate dehydrogenase (G6PDH) in solutions exists as the equilibrium mixture of dimers and tetramers. Only dimers are active. The equation resp onding to these conditions was used to determine the parameters: the sp ecific activity of dimers and the disso ciation equilibrium constant of tetramers. The stability of G6PDH dimers were shown to b e maintained due to the conformational lo ck, a multiple-p oint intersubunit contact in the hydrophobic region of "cylindrical" protomers. The conformational lo ck is formed by two isolated contacts at least. In the consecutive mechanism of G6PDH thermal inactivation, the destruction of one of these contacts results in a labile dimer. The latter disso ciates to inactive monomers. At 46 C, the equilibrium constant of this pro cess II Kdis = (6.2 ± 0.4) nM, whereas the sp ecific activity dimers a2 = (13 ± 0.2) в 106 mol NADPH/min.mol of active sites. The kinetic analysis showed that the inactivation process is preceded by tree steps: the disso ciation of tetramers to active stable dimers, the emergence of labile dimers and their disso ciation to inactive protomers. The final step of G6PDH thermal inactivation is an irreversible inactivation of protomers, the denaturation with the rate constant k = 6 в 10-3 min -1 .

G6PDH is glucose-6-phosphate dehydrogenase (Dglucose-6-phosphate, oxidoreductase) belongs to one of the most important and vast class of enzymes NAD-dependent dehydrogenases. G6PDH catalyzes dehydration of glucose6-phosphate (G6P) to 6-phosphategluconic lactone. As hydride-ion acceptor, the enzyme uses NADP + . G6PDH amino acid composition was totally sequenced in 1969 [1]. A typical feature of G6PDH, like of other dehydrogenases, is its inactivation resulting from its dissociation [2, 3]. Not a single dehydrogenase was shown to have an active monomer. This is indicative of the importance of subunit contacts for catalytic properties to be pronounced. G6PDH from beer yeast have been most studied [2]. The method of sedimentation equilibrium was used to establish that G6PDH exists as monomer, dimer and tetramer entities. The molecular mass of the dimer is 102 kD. The second substrate of the reaction, NADP + , affects the 2 dimers 1 tetramer equilibrium in G6PDH solutions. The dimeric entities stable in a wide pH range and even in very diluted solutions, which is definitely defined by a fair reliability of the conformational lock stabilizing G6PDH dimer. The presence of NADP + and NADPH, unlike the second substrate G6P, causes the association of the active dimeric form of the enzyme to the tetrameric entity [3]. The work shows G6PDH dimeric form to contain two equivalent centers of NADP + binding with the dissociation constants K1 = 45 µ M and K2 = 13 µ M (pH 8.0, 27 C). Each dimer contains a hydrophobic region between the subunits-conformational lock, the distortion of which entails the dissociation of the active dimers to inactive monomers [3]. The same work reported that G6PDH monomers have a cylindrical shape 68 в 34 ° in size. A A detailed kinetic analysis for yeast G6PDH was reported [2, 4, 5]. The kinetics of the enzyme action obeys

the Michaelis-Menten Law with an independent binding of substrates. At pH 8­9, KG6P = 3.5 в 10-5 M and KNADP = 4 в 10-6 M. NADP + analogs act on G6PDH thermal stability variously, which is explicable by various character of conformational changes upon binding of the analogs [6]. Hence, the analogs affect the reliability of the conformational lock and the capacity of the dimers to form tetramers. It was reported earlier [7] that at low temperatures, G6PDH is inactivated by the consecutive dissociation mechanism: E
4 k2 2E k
-2

2

k1 k
-1

4E

1

kd - 4E d .

(1)

inactive

active

inactive

I II Here Kdis = kk22 and Kdis = kk11 . - - The task of this research is to offer additional experimental results to confirm mechanism (1) and to kinetically analyze the stability of the conformational lock in G6PDH dimer.

Materials and Metho ds Materials used: crystalline G6PDH extracted from yeast was a suspension in 3.2 M ammonium sulfate (Ferak, Berlin); NADP + monosodium salt and magnesium chloride (Russian production). The starting rate of the reaction was determined from kinetic curves for accumulation of NADPH (reaction product) at 340 nm on a spectrophotometer (Hitachi) in thermostatted cuvettes. The composition of the overall reaction mixture (2 ml) was 1.1 ml 0.2 M borate buffer, pH 8.0 + 0.4 ml enzyme of relevant concentration + 0.5 ml mixture of substrates containing

Chair of Chemical Enzymology, Department of Physical Chemistry, Faculty of Chemistry, The Lomonosov Moscow State University.


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VESTNIK MOSKOVSKOGO UNIVERSITETA. KHIMIYA. 2000. Vol. 41, No. 6. Supplement

9.4в10-4 M G6P, 1.44в10-3 M NADP + and 7.5в10-3 M MgCl 2 . Thermal inactivation was conducted in thermostatted vessels at 45­46 C. In certain time intervals, aliquots (0.4 ml) of this mixture were taken to measure the enzymatic activity.

dimeric form. From this, at any [E ]0 , b 1 . This curve has a maximum and describes process E
4 k2 2E k
-2

2

k1 k
-1

4E1 . inactive

(3)

inactive

active

Results and Discussion It was reported earlier [3] that G6PDH is an equilibrium mixture E4 2E2 , tetramers being inactive. The specific catalytic activity as a function of protein concentration in solution is a declining curve in the concentration range [E0 ] 0.5­18 nM (Fig. 1). It is notably simpler to define the association constant from the concentration dependence of the specific activity when one of the entities is inactive. At a4 = 0 (with inactive tetramers), expression 1 1 2Kass = +2 a a2 a2 A (2)

According to scheme (3), the intermediate active entity is dimer. At the first step of the process, the dissociation of tetramers increases the activity but emergence of inactive monomers entails its decrease. This fact hinders to calcuI late Kdis at 46 C. In this case, another procedure turns out useful: to represent the experimental results obtained at various initial protein concentrations in the coordinates of linear equation 1 1 1-b = + 2 [E ]0 b 2bmax Kdis
max

,

(4)

holds. Here A and a are the overall and specific activities, respectively; a2 is the specific activity of dimers. The experimental results should be linearized in the coordinates 1/a, A . Using relationship (2), the obtained results were treated to derive the necessary parameters tabulated below.

where bmax is the value of b in the maximum point; is the ratio of the constants k2 /k1 (scheme (3)). Thus derived I Kdis value is given in the table. The method of approximated calculations supposes to define the ratio of the constants from the length of the intercept cut off on Y -axis. It was reported [7] that the dimeric entity of the protein in solution dissociates further. For this reason, it is possible to define the k2 /k1 value for test system, where k2 is the rate constant for decomposition of inactive tetramers to active dimers whereas k1 is the rate constant for decomposition of active dimers to inactive monomers. According to the kinetic analysis thus conducted, = k2 /k1 = 14.3 . The value I of k1 was derived in the same conditions at [E ]0 Kdis , i. e., in absence of tetramers. In this case, the kinetics of thermal inactivation is described by a declining curve which has a knee in semilogarithmic coordinates. The starting region of the curve is linearized in the coordinates of equation (5). F ( ) = 2 1 - 0 2 0
2

=

3 - k1 t. 2

(5)

Fig. 1. Dynamics of G6PD relative activity after its thermal inactivation in 0.2 M borate buffer, pH 8.0, at 46 C. [E ]0 = 23 nM. Table 1 Kinetic parameters of G6PDH disso ciative pro cesses at various temp eratures in 0.2M b orate buffer, pH 8 t, C 20 46 K
I dis

, nM

II Kdis , nM

a2 , mol NADPH/min.mol G6PDH (9.5 ± 1) в 10-5 (13 ± 0.1) в 10-6

0.71 ± 0.95 24 ± 3

-- 6 .2 ± 0 .4

The kinetic curves of thermal inactivation in the coordinates "relative activity ( b )--time ( t )" have a maximum each. The physical sense of ( b ) value is simple: this is the ratio of the observed enzymatic activity for the initial protein concentration E0 to the enzymatic activity for the same concentration provided all protein is in the active

Neglecting the temperature difference 1 C at the known value of k1 (45 C) = 0.035 min -1 , it is easy to derive k2 (46 C) = 0.5 min -1 . The computed analysis of I II scheme (3) showed that at Kdis = 10Kdis and at the selected values = 20 , 10, 5, and 1, respectively; the maximum appears only at > 5 whereas at 1 the maxima were not observed on the kinetic curves. This analysis shows that when the constants are equal, it is impossible to kinetically discriminate two consecutive processes in scheme (3). Yet, the number of steps preceding the inactivation step is definable from the kinetic curves /0 В 1 (Fig. 2) on having drawn a tangent at the knee to its cross with Y -axis at the point R = + 1 . The value is related to the number of steps n through empirical expression as follows [8], 0.13 + n= . (6) 0.13 - 0.05 Figure 2 shows that all tangents cross in one point R = 1.22 . From this, derive = 0.22 , which corresponds to the minimal number of the ulterior steps n = 3 . That is in scheme (1), there is an additional step of conversion of


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129
I II eters in scheme (1): Kdis , Kdis , k1 , k2 , and kd . At I II 46 C, Kdis = (24 ± 3) nM whereas Kdis = (6.2 ± 0.4) nM. This implies that at operative concentrations of the enzyme 1­19 nM and at this temperature, the concentration of tetramers can be neglected. The rate constant for dissociation of tetramers k2 = 0.5 min -1 , which is higher by a factor than the rate constants of labile dimers k1 = 0.035 min -1 whereas the rate constant of the irreversible inactivation (denaturation kd = 6 в 10-3 min -1 . The stability of G6PDH dimers is provided by the conformational lock formed, at least, by two independent intersubunit contacts in the hydrophobic region of protomers. A similar structure of the conformational lock was disclosed kinetically and proved by structural studies for the dimer of alkaline phosphatase from E. coli [9].

Fig. 2. Determination of the dimensionless relative lag p eriod = R - 1 from kinetic curves of G6PD thermal inactivation in 0.2 M b orate buffer, pH 8.0, at 46 C. [E ]0 = 1 nM (1); 6 nM (2); 9 nM (3), and 19 nM (4).

References 1. Engel H.J., Domschke W., and Alberti G.F. Biochem. Biophys. Acta. 1969. 191. P. 509. 2. Yue R.H., Noltman E.A., and Kuby S.A. J. Biol. Chem. 1969. 244. P. 1353. 3. Wrigly N.G., Heather J.V., Bonsignore A., and De Flora A. J. Mol. Biol. 1972. 68. P. 483. 4. Kurganov B.I. Molekulyarnaya Biologiya. 1967. 1. P. 17. 5. Luzzatto L. Biochem. Biophys. Acta. 1967. 146. P. 18. 6. Mattiasson B. and Mosbach K. Biochem. Biophys. Acta. 1971. 235. P. 253. 7. Zaitzeva E.A., Chukhrai E.S., and Poltorak O.M. Modern enzymology: problems and trends. Eds. Kurganov B.I., Kochetkov S.N., and Tishkov V.I. Nova Science publishers. L.-NY. 1995. P. 585. 8. Bednarek P.Z., Kaberdin V.R., Taschlickyi V.N., Poltorak O.M., and Chukhrai E.S. Vestn. Mosk. Univ. Khimiya. 1987. 28. P. 523. 9. Poltorak O.M., Chukhrai E.S., Torshin I.Y., Atiaksheva L.F., Trevan M.D., and Chaplin M.F. J. Mol. Cat. B: Enzymatic. 1999. 7. P. 165.

the active stable dimer to the active labile dimer capable of dissociation.
I E4 2E 2stab II 2E 2lab III - 4E1 .

(7)

From this, it follows that G6PDH dimer is fixed with two multiple-point contacts. Destruction of one of them entails the emergence of less stable isomer E2 . The conformational lock is located in the hydrophobic region of the cylindrical molecule of G6PDH protomer [3]. Evidently, the dimer is stabilized due to the presence of the conformational lock and the formation of tetramers which can be considered as a reserve for the native enzyme. The amount of this reserve is regulated by the second substrate of NADPH and its analogs [6]. Thus, this work has successfully showed the procedure for G6PDH stabilization and kinetically described the mechanism of the low-temperature inactivation of the enzyme, i. e., derived all kinetic param-