TY - JOUR
T1 - Kinetic Behavior of Alkaline Phosphatase-Collodion Membranes
AU - Goldman, Rachel
AU - Kedem, Ora
AU - Katchalski, Ephraim
PY - 1971/1/1
Y1 - 1971/1/1
N2 - A theoretical analysis of the role of the Nernst diffusion layer in determining the apparent kinetic behavior of an enzyme membrane is given. It is shown that the overall rate of reaction, V, is determined by the concentration of substrate at the membrane-solution interface, S0, and the catalytic and physical parameters of the enzyme-membrane. The value of S0 is smaller than that of the concentration of substrate within the bulk of the solution, Sb, and is determined by the thickness of the diffusion layer as well as the catalytic parameters of the enzyme-membrane-substrate system. Quantitative relations between V and S0 were derived for enzyme-membrane catalysis in the absence and presence of product inhibition. Two enzyme-membrane-substrate systems were analyzed: an alkaline phosphatase membrane acting on p-nitrophenyl phosphate and a papain membrane acting on benzoyl-L-argininamide. The effect of diffusion layers 10- and 50-μ thick was shown to depend primarily on the values of V and that of the apparent Michaelis constant, Km(app), for the enzyme-substrate couple. The contribution of the diffusion layer in determining the kinetic behavior of enzyme-membranes was demonstrated experimentally with alkaline phosphatase-collodion membranes. Three-layer alkaline phosphatase-collodion membranes, in which the enzyme layers were 1.5-, 3.0-, and 9.0-μ thick, were prepared by controlling the time of enzyme adsorption by a collodion membrane 210-μ thick. The adsorbed enzyme was less stable at elevated temperatures (60 and 80°) than the native enzyme, but could be stored at 4°, for at least 14 days without detectable loss in activity. The pH-activity profile of the alkaline phosphatase membranes, within the range of pH 8-11, using p-nitrophenyl phosphate as substrate, at low and high ionic strength, but in the absence of buffer, deviated from that recorded for the native enzyme. The deviation could be accounted for by assuming a local pH within the membrane of 1-2 pH units lower than that of the external solution. The above deviation was cancelled in the presence of 0.4 M borate buffer. A plot of the experimental values of V vs. Sb for the three alkaline phosphatase-collodion membranes prepared, showed that the Km'(app) values obtained (assuming Km'(app) = Sb at V/Vmax = 0.5) are higher by a factor of 10 than those calculated for the corresponding membranes taking product inhibition into account and ignoring the presence of a diffusion layer. The apparent Michaelis constants determined experimentally were 25- to 350-fold that of the native enzyme. The above deviations could be accounted for by assuming values of 42-66 μ for the thickness of the diffusion layer adhering to the enzyme-membranes employed.
AB - A theoretical analysis of the role of the Nernst diffusion layer in determining the apparent kinetic behavior of an enzyme membrane is given. It is shown that the overall rate of reaction, V, is determined by the concentration of substrate at the membrane-solution interface, S0, and the catalytic and physical parameters of the enzyme-membrane. The value of S0 is smaller than that of the concentration of substrate within the bulk of the solution, Sb, and is determined by the thickness of the diffusion layer as well as the catalytic parameters of the enzyme-membrane-substrate system. Quantitative relations between V and S0 were derived for enzyme-membrane catalysis in the absence and presence of product inhibition. Two enzyme-membrane-substrate systems were analyzed: an alkaline phosphatase membrane acting on p-nitrophenyl phosphate and a papain membrane acting on benzoyl-L-argininamide. The effect of diffusion layers 10- and 50-μ thick was shown to depend primarily on the values of V and that of the apparent Michaelis constant, Km(app), for the enzyme-substrate couple. The contribution of the diffusion layer in determining the kinetic behavior of enzyme-membranes was demonstrated experimentally with alkaline phosphatase-collodion membranes. Three-layer alkaline phosphatase-collodion membranes, in which the enzyme layers were 1.5-, 3.0-, and 9.0-μ thick, were prepared by controlling the time of enzyme adsorption by a collodion membrane 210-μ thick. The adsorbed enzyme was less stable at elevated temperatures (60 and 80°) than the native enzyme, but could be stored at 4°, for at least 14 days without detectable loss in activity. The pH-activity profile of the alkaline phosphatase membranes, within the range of pH 8-11, using p-nitrophenyl phosphate as substrate, at low and high ionic strength, but in the absence of buffer, deviated from that recorded for the native enzyme. The deviation could be accounted for by assuming a local pH within the membrane of 1-2 pH units lower than that of the external solution. The above deviation was cancelled in the presence of 0.4 M borate buffer. A plot of the experimental values of V vs. Sb for the three alkaline phosphatase-collodion membranes prepared, showed that the Km'(app) values obtained (assuming Km'(app) = Sb at V/Vmax = 0.5) are higher by a factor of 10 than those calculated for the corresponding membranes taking product inhibition into account and ignoring the presence of a diffusion layer. The apparent Michaelis constants determined experimentally were 25- to 350-fold that of the native enzyme. The above deviations could be accounted for by assuming values of 42-66 μ for the thickness of the diffusion layer adhering to the enzyme-membranes employed.
UR - http://www.scopus.com/inward/record.url?scp=0015210986&partnerID=8YFLogxK
U2 - 10.1021/bi00777a024
DO - 10.1021/bi00777a024
M3 - Article
C2 - 5538604
AN - SCOPUS:0015210986
SN - 0006-2960
VL - 10
SP - 165
EP - 172
JO - Biochemistry
JF - Biochemistry
IS - 1
ER -