The study of pectin methylesterase and wall-loosening enzyme activities in situ, as well as the estimation of the electrostatic potential of the cell wall, suggest a coherent picture of the role played by metal ions and pH in cell-wall extension. Cell-wall growth brings about a decrease of local proton concentration because the electrostatic potential difference (delta psi) of the wall decreases. This in turn activates pectin methylesterase, which restores the initial delta psi value. This process is amplified by the attraction of metal ions in the polyanionic cell-wall matrix. The amplification process is basically due to the release of enzyme molecules that were initially bound to 'blocks' of carboxy groups. This increase of metal-ion concentration also results in the activation of wall-loosening enzymes. Moreover, the apparent 'inhibition' of pectin methylesterase by high salt concentrations may be considered as a device which prevents the electrostatic potential from becoming too high.
The pectin methyl esterase from soybean cell walls has been isolated and purified to homogeneity. It is a protein with a relative molecular mass close to 33000. The enzyme is maximally active at a pH close to 8 and its pH dependence may be explained by a classical Dixon model, where the two interconvertible enzyme ionization states coexist.The outflux of protons from cell walls, upon raising the ionic strength, may be taken as an indirect estimate of the fixed charge density. If the cell-wall fragments are pre-incubated at pH values between 5 and 9, the outflux of protons rises with the pH of pre-incubation. This implies, as postulated from the theory developed in the preceding paper, that alkaline pH favours the activity of pectin methyl esterase and that this enzyme effectively generates the fixed negative charges of the cell wall. Therefore the pectin methyl esterase reaction builds up the Donnan potential, dy, at the cell surface.The cell-wall charge density, estimated from the proton outflux, as well as from the titration of methyl groups on the cell wall, reaches a maximum between the third and the fourth day of growth. While the cell-wall volume increases and reaches a plateau, the fixed charge density increases at first and then declines. This is understandable if one assumes that the building up of a high charge density is a co-operative phenomenon and that the local pH inside the wall rises during cell growth. When both the cell-wall volume and the charge density increase together, this suggests that the local pH inside the wall lies within the critical pH range associated with the steep response of the system. When the cell-wall volume increases together with a decrease of the fixed charge density, the local pH should have dropped below this critical pH range. Under these conditions the pectin methyl esterase remains inactive, or poorly active.As the number of fixed negative charges increases, calcium becomes tightly bound to cell walls. This binding is so tight that the net charge density is minimum when the calcium concentration is maximum.The experimental results, presented above, offer experimental support to two important ideas discussed in the preceding paper, namely that pectin methyl esterase reaction builds up the Donnan potential at the cell surface, and that this response may be co-operative with respect to pH.During the plant growth, pectic compounds are integrated in the cell wall as uncharged, methylated molecules. Pectin methyl esterase is believed to de-esterify these compounds and to generate the fixed negative charges that result in the building up of a Donnan potential between the inside and the outside of the cell wall matrix [l -51. Pectin methyl esterase has been isolated and purified from fruits [6, 71 and from Vigna hypocotyls [S]. In this latter case at least, the enzyme has been shown to be bound to the cell walls and to have an optimum pH close to 8. Moreover, there appears to be a correlation between the activity of this enzyme and plant cell growth [8, 91. However pectin m...
The ionic species ATP*-and Mg2+, a t high concentrations, decrease the rate of glucose phosphorylation catalyzed by yeast hexokinase. A theoretical study of all the possible mechanisms (271 models) of this inhibition has been made. The comparison of these mechanisms with the experimental results shows that only one model can explain the inhibition of the reaction by ATP, and that only one can also explain the effect of the high magnesium concentrations.It was thus possible to show that the ionic species ATP4-can be bound on hexokinase and on the hexokinase-glucose complex. Its affinity is much stronger for the second form of the enzyme than for the first. The complexes thus formed are devoid of any activity ("dead-end" complexes). The inhibition of the hexokinase by the high nucleotide concentrations is therefore due to the formation of these complexes. A ternary chelate of the Mg(ATP)z-type does not exist.Magnesium possesses no affinity for the enzyme, Its role in the catalysis must therefore be indirect. The results presented agree with the idea that the metal polarizes the phosphoryl bond of the ATP that is split when glucose-6-phosphate is formed. Magnesium can form with ATP a ternary chelate of the Mg,ATP type, unable to be bound on the hexokinase. The inhibiting effect of high concentrations of the metal can be explained by the formation of this inactive ternary chelate and by the decrease in concentration of the MgATP2-complex.I n two recent articles [1,2] we presented the idea that glucose phosphorylation is effected following the ordered mechanism :in which E represents the enzyme, G the glucose, MA and MA' the chelates MgATP2-and MgADP-, G' the glucose-6-phosphate. The rate constants involved in this mechanism were determined [l] and the kinetics of the formation and decomposition during the pre steady-state period, of the various enzyme-substrates and enzyme-products complexes analyzed using an analog computer [3].The results obtained and the above interpretation were recently criticized by Fromm [4] who maintains that the hexokinase mechanism is of a random type. According to this investigator, the enzyme binds glucose and the chelate MgATP2-equally well. These criticisms and this affirmation seem invalid to us. I n fact, Colowick [S] and our group [B] have recently shown, using equilibrium dialysis techniques, that hexokinase binds glucose, but is practically unable to form a complex with the nucleotide in the absence of hexose. This result, in agreement with the ordered model (l), seems to rule out the random mechanism [4].However, there is no doubt that the model (1) is too simplified to explain all the types of interactions that can exist between the enzyme and its substrates. Thus the low ATPase activity of hexokinase [7,8] shows that ATP can be bound, very slightly, on the enzyme. Moreover Hammes [9] proposed the idea that the ionic species ATP4-possesses a low but real affinity €or the glucose-hexokinase complex. Also, the studies of Fromm [lo] and Rose [ll] showed that AMP and adenine beha...
Thirty-one different models corresponding to all plausible mechanisms of glucose phosphorylation conditioned by yeast hexokinase have been analysed. Comparison of these models with the results given by kinetic study of the enzyme has enabled us to prove that the catalysis corresponds to an ordered mechanism in which hexokinase binds first glucose and then the chelate MgATP2-. The latter substrate is not able, to any great extent, to form directly the first complex with the phosphotransferase. The ionic species Mg2+ and ATP4-have only a negligible affinity for the enzyme, or the enzyme-glucose complex.Glucose-6-phosphate and ADP appear in the medium following the decomposition of the enzyme-glucose-magnesium-ATP complex. This decomposition is also controlled by an ordered mechanism, MgADP-being formed first, and glucose-6-phosphate afterwards. MgADP-is able not only to fix itself on the enzyme-glucose-6-phosphate complex but also on the enzyme-glucose complex.This mechanism is in contradiction with that of Fromm. On the other hand it agrees with that postulated but not proved by Hammes and Kochavi. The rate constant values in the proposed mechanism have been calculated. They are different from those calculated by Hammes and Kochavi.Recent kinetic studies of hexokinases have led to contradictory results. Even though some workers in this field [l-31 admit that the catalysis is mediated through an enzyme-phosphate complex, it seems that this conception is not widely accepted, a t least as far as yeast hexokinase is concerned. Most authors now agree that glucose phosphorylation involves a complex of the enzyme-glucose-magnesium-ATP type. However, the mechanism of the catalysis still remains very much a matter of controversy. Hammes and Kochavi [4-61 reached the conclusion that yeast hexokinase binds glucose first, and then the chelate magnesium-ATP. On the other hand, Toews [7] claims that the enzyme, from another source it is true, fixes MgATP2-before hexose. I n both cases the mechanism of the glucose phosphorylation would be an ordered one ("Bi Bi Mechanism" in Cleland's terminology [S]). Fromm and his colleagues [9, lo], on the contrary, consider they have shown, with yeast hexokinase, the existence of a rapid equilibrium random mechanism among all the possible enzymesubstrate complexes. Enzyme could then bind glucose and magnesium-ATP in either order.I n fact, to be able to assert that a model is valid, one has not only to prove that the experimental results agree with the model, but also that they disagree with all the other apparently plausible models. This latter condition does not seem to have always been entirely satisfied, a t least as far as the kinetic study of hexokinase is concerned. Thus, for example, Hammes [4,5] concluded from his results the existence of an ordered mechanism, and that, a few years later, Ottolenghi [ill showed that these same results could also be interpreted by a rapid equilibrium random mechanism of the type proposed by Fromm [9,10].The study of the phosphotransferase catalytic m...
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