TY - JOUR
T1 - Fumarase
T2 - Viscosity dependence of the kinetic parameters
AU - Sweet, William L.
AU - Blanchard, John S.
N1 - Funding Information:
1 This work was supported by NIH Grant ander and Alexandrine Sinsheimer Scholar * Author to whom correspondence should
PY - 1990/2/15
Y1 - 1990/2/15
N2 - Fumarase catalyzes the reversible, stereospecific hydration of fumarate to form l-malate. We have determined the viscosity dependence of V K and V in both the forward and the reverse directions at pH 6.9 in the absence and presence of several viscosogenic reagents. V K for fumarate hydration decreases with increasing concentrations of glycerol and sucrose, but is unaffected by increasing concentrations of the polymeric viscosogen polyethyleneglycol (av MW, 10,000 da). V K for malate dehydration similarly decreases with increasing concentrations of both glycerol and sucrose, but is unaffected by increasing concentrations of polyethylene glycol. Equilibrium constants, calculated from the ratio of V K values for malate dehydration and fumarate hydration at various concentrations of glycerol, closely match the experimentally determined equilibrium constants at the same concentrations of glycerol. Both experimental and calculated equilibrium constants decrease with increasing concentrations of viscosogens. V K for the dehydration of (-)-tartrate, a poor substrate, is unaffected by increasing concentrations of glycerol. Analysis of the microviscosity dependence of malate dehydration and fumarate hydration suggests that both substrates bind at diffusion-limited rates. The viscosity dependence of substrate and product dissociation steps may also contribute to the viscosity dependence of V K values for both substrates. The viscosity dependence of the maximal velocities argues that product dissociation steps are rate-limiting and diffusion controlled.
AB - Fumarase catalyzes the reversible, stereospecific hydration of fumarate to form l-malate. We have determined the viscosity dependence of V K and V in both the forward and the reverse directions at pH 6.9 in the absence and presence of several viscosogenic reagents. V K for fumarate hydration decreases with increasing concentrations of glycerol and sucrose, but is unaffected by increasing concentrations of the polymeric viscosogen polyethyleneglycol (av MW, 10,000 da). V K for malate dehydration similarly decreases with increasing concentrations of both glycerol and sucrose, but is unaffected by increasing concentrations of polyethylene glycol. Equilibrium constants, calculated from the ratio of V K values for malate dehydration and fumarate hydration at various concentrations of glycerol, closely match the experimentally determined equilibrium constants at the same concentrations of glycerol. Both experimental and calculated equilibrium constants decrease with increasing concentrations of viscosogens. V K for the dehydration of (-)-tartrate, a poor substrate, is unaffected by increasing concentrations of glycerol. Analysis of the microviscosity dependence of malate dehydration and fumarate hydration suggests that both substrates bind at diffusion-limited rates. The viscosity dependence of substrate and product dissociation steps may also contribute to the viscosity dependence of V K values for both substrates. The viscosity dependence of the maximal velocities argues that product dissociation steps are rate-limiting and diffusion controlled.
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U2 - 10.1016/0003-9861(90)90569-K
DO - 10.1016/0003-9861(90)90569-K
M3 - Article
C2 - 2306120
AN - SCOPUS:0025174784
SN - 0003-9861
VL - 277
SP - 196
EP - 202
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 1
ER -