Resistance to inactivation by EGTA of the enzyme-substrate and enzyme-phosphate complexes of alkaline phosphatase

Abstract

Bovine intestinal mucosal alkaline phosphatase is inactivated by the chelating agent EGTA. Several concentrations of the enzyme were incubated with EGTA and a range of concentrations of the substrate p-nitrophenyl phosphate to determine the substrate concentration as a function of time. As predicted by a recently developed theory [Duggleby (1986) J. Theor. Biol. 123, 67-80], catalysis ceases before all substrate is exhausted. An analysis of these final substrate concentrations according to the theory revealed that, whereas the free enzyme is unstable, the effect of EGTA is counteracted when either the substrate or product (phosphate) is bound. Comparison of the results with those obtained by direct stability measurements and steady-state kinetic experiments gave a qualitatively and quantitatively consistent body of evidence in support of this interpretation.

Aggregation and inhibition of rat intestinal alkaline phosphatase by high concentrations of calcium. Reversibility of the processes

“…As a consequence one could conclude that EGTA did not chelate the zinc that is in the structure of IAP. Other works have proved that chelation of zinc that is in the structure of the enzyme is dependent of the presence of substrates [17].…”

Kinetic and stability studies on the chloroperoxidase complexes in presence of tert-butyl hydroperoxide

“…Duggleby suggested a graphical method, the J plot, for the determination of the inactivation rate constants from the final substrate concentration. A kinetic analysis has been made for the case in which either the free enzyme or the two types of enzyme-substrate complexes are unstable, spontaneously or as a result of the addition of an oxidant, respectively; the explicit time course equations of all of the species involved have been derived [16][17][18] assuming that, during the time considered, the oxidant concentration remains approximately constant. In the usual enzymatic cycle of peroxidases, the native enzyme reacts with an oxidant to form intermediate compound I with two oxidising equivalents above ferric state; compound I is then reconverted to the native form via one to two electron steps in which two reducing substrate molecules are oxidized into radicals [19].…”

Kinetics of inactivation of bovine pancreatic ribonuclease A by bromopyruvic acid

“…The kinetics of enzyme modification and denaturation have been treated in two different ways, depending on whether a significant proportion of substrate is consumed in the enzymecatalysed reaction [2][3][4][5] or whether the substrate concentration can be considered to be essentially constant during the period of observation and hence set equal to its initial value in the derivation of integrated rate expressions describing the time dependence of product formation [1,6]. The latter approach has been extended beyond the treatment of relatively simple cases of irreversible modification of an enzyme catalysing a singleintermediate one-substrate reaction to include more complicated models [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23].…”

“…The non-competitive inhibition type indicates that neither the substrate nor the product affects the binding of bromopyruvic acid to the enzyme and that the ionization state of His-119 may be the same in both the enzyme-substrate and the enzyme-product complexes. stability of alkaline phosphatase during inactivation by EGTA [4].…”