B5 Influence of Multivalent Cations on Kinetics of a Cell Wall Enzyme

Plant cell walls contain many ionizable groups and may be regarded as immobilized polyelectrolytes. The ionic behavior of cell walls has been described by the well-known theories of Donnan and Gouy-Chapman.

The enzyme acid phosphatase, when bound to cell walls but not when solubilized in solution, is activated by increasing the ionic strength of the reaction mixture [1]. This apparent enzyme activation may be attributed to a decrease of the Donnan potential, which can inhibit the accessibility of negatively charged substrates to the cell walls. Acid phosphatase in plant cell walls may be important in hydrolyzing and solubilizing organic phosphate-containing macromolecules in the soil independent of microbial activity.

Dehydrogenase Enzyme Assay For Soil
Figure 6.8 pH dependence of the activity of isocitrate dehydrogenase. Points are experimental data; straight line is best fit by linear regression. (The authors thank Dr. H. M. Farrell for the original data.)

The influence of multivalent cations on the activity of both bound and solubilized acid phosphatase associated with the primary cell walls of potato tubers and corn roots has been investigated. The results were analyzed in terms of binding equilibria using the thermodynamic linkage approach [1], It was assumed that the observed change of acid phosphatase activity is due to a certain unique binding of cations to sites consisting of components of cell walls.

The analysis of the enzyme activity vs. cation concentration data assumed that molecules of acid phosphatase are uniformly distributed among many equivalent subdomains in the structure of the cell walls. Binding n metal ions to the subdomain, either directly to the enzyme, to its immediate environment, or to both, will cause a concentration-dependent change in the activity of the enzyme.

Table 6.9 Model for a Peak-Shaped Dependence of Enzyme Activity on pH

Assumptions: Two protonation equilibria as in eqs. (6.45) and (6.46) Regression equations:

Regression parameters: Data:

For the simplest case, consider the following binding equilibrium between the subdomain (M) containing the enzyme and metal ions (X):

in which MXn and M represent the domains with and without bound metal ions, respectively. Ka is the association equilibrium constant. These equations are the same as the simple overall equilibrium expressions in eqs. (6.3) and (6.4).

The dependence on metal ion concentration of the rate of enzymic hydrolysis of p-nitrophenyl phosphate (PNP-P) by cell wall acid phosphatase has been examined. By keeping the substrate concentration in large excess over the Michaelis constant KM (i.e., KM = 1 IKa), the observed apparent enzyme activity, Aapp, for the hydrolysis of PNP-P in the presence of test cations, is expressed as

where A, is the activity of M and A2 is the activity of MX„. The fractions fM,0 and fM i are given in Table 6.1.

A2 may be either greater or smaller than Aj. This can be expressed as

Q is greater than 1 for enhancement of the activity and smaller than 1 for inhibition of the activity. The values of A\ and C can be easily estimated from the activity vs. metal ion concentration data using the model in Table 6.10.

Plant cell walls contain relatively small amounts of protein, about 10% of the total dry weight of cell walls. Even assuming that all the protein in the cell walls is acid phosphatase, the maximum enzyme content would be very much smaller than the total concentration of metal ions (Jf) added

Table 6.10 Single Binding Site Model for Activity of Acid Phosphatase in the Presence of Metal Ions (X)

Assumptions: Domains of activity are uniformly distributed in cell wall. Cx > Ci,, so that [X] = Cx

Regression equation:

A ,4, QA{KaCx app l + KaC"x l + K,C"X Regression parameters: Data:

Special instructions: Run a series of nonlinear regressions with successively increasing fixed integer n values beginning with n = 1 until the best standard deviation of regression is achieved in the experiments. Hence the substitution [X] = Cx is fully justified. The model based on eqs. (6.48), (6.49), and the preceding assumptions is given in Table 6.10.

The results of this analysis for cell wall acid phosphatase in the presence of several heavy metal ions are illustrated in Figure 6.9. The binding parameters are summarized in Table 6.11. In most cases, the best fits to the data were obtained by the use of the model in Table 6.10.

However, for Hg2+-binding to potato cell wall, the model in Table 6.10 did not fit the data. The activity vs. [Hg2+] plot shown in Figure 6.9 indicated that the binding of Hg2+ at low concentrations enhances the activity. At higher concentrations of Hg2+, inhibition becomes predominant. A model to account for this observation employed two independent binding sites for Hg2+ in the subdomains of the potato cell wall. Addition of n moles of X per mole of binding sites enhances the activity, whereas binding of

Figure 6.9 Influence of multivalent cations on the relative acid phosphatase activity of potato cell wall, (a) Mg2+; (b) Al3+; (c) Hg2+. Points are experimental data and lines are best fits onto models discussed in text (adapted with permission from [1].)

Concentration (mM)

Figure 6.9 Influence of multivalent cations on the relative acid phosphatase activity of potato cell wall, (a) Mg2+; (b) Al3+; (c) Hg2+. Points are experimental data and lines are best fits onto models discussed in text (adapted with permission from [1].)

Table 6.11 Metal Ion Binding Parameters for Cell Wall Acid Phosphatase"

Source

Ion

Ka, mM

n

RSD, %h

Effect

Potato

Mg2+

3.78

1

15

Stimulation

Potato

Al3+

1.40

2

4

Inhibition

Potato^

Hg2+

108.4

1

3

Stimulation

Potato^

(Second K)c

1.4 x 104

4

Inhibition

Corn

Hg2+

4.82

1

2

Inhibition

a Best fit model in Table 6.10 unless otherwise noted.

b RSD refers to the average deviation between observed and calculated values of A. c Two-site model in Table 6.11 for the effect of Hg on potato cell wall acid phosphatase.

a Best fit model in Table 6.10 unless otherwise noted.

b RSD refers to the average deviation between observed and calculated values of A. c Two-site model in Table 6.11 for the effect of Hg on potato cell wall acid phosphatase.

an additional m moles of X inhibits the reactions. This binding model is represented as

The observed activity, A, can then be expressed as

where /M2 = fraction of M as XmMXn or XnMXm and A3 = Q'AU with Q' < 1 to account for the inhibition at higher [Hg2+], This two-site model is described in Table 6.12.

Using the two-site model again necessitates searching for the integer values of n and m that give the lowest standard deviation of regression. The data for Hg2+ and potato cell wall acid phosphatase were best fit with n = 1 and m = 4. On the other hand, a sequential binding model that

Table 6.12 Two Binding Site Model for Activity of Acid Phosphatase in the Presence of Metal Ions (*)

Assumptions: Domains of activity are uniformly distributed in cell wall.

Regression equation:

. _At + QAiKaCx + Q'Ax(KaCx + KmKnCrn) app 1 + K„C"X + KmC% + KmK„Cx*"

Regression parameters: Data:

Special instructions: Run a series of nonlinear regressions with successively increasing fixed integer n values and m values until the best standard deviation of regression is achieved specified that the binding of the n sites occurred before the m sites did not provide a satisfactory fit to these data.

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