J. Biol. Chem., Vol. 261, Issue 18, 8163-8166, 06, 1986
The determination of enzyme-substrate dissociation rates by dynamic isotope exchange enhancement experiments
SC Kim and FM Raushel
A new method for the determination of dissociation rates of enzyme-
substrate complexes has been developed. The rate of exchange of a labeled
product back into the substrate is measured during catalysis of the forward
reaction when the forward reaction is kept far from equilibrium by the
enzymatic removal of the nonexchanging product. The ratio of the exchange
rate and the net rate for product formation is then determined at various
concentrations of the exchanging product. A plot of this ratio is a
diagnostic indication of the kinetic mechanism and the relative rates of
product dissociation from the binary and ternary enzyme complexes. This
technique has been applied to the reaction catalyzed by bovine liver
argininosuccinate lyase. The ratio for the rate of exchange of fumarate
into argininosuccinate and the net rate for product formation was found to
increase with the concentration of fumarate but to reach a limit of 3.3.
The ratio of rates was half- maximal at 36 mM fumarate. The data have been
interpreted to indicate the argininosuccinate lyase has a random kinetic
mechanism. The calculated lower limit for the rate of release of arginine
from the enzyme-fumarate-arginine complex is 0.35 times as fast as the Vmax
in the reverse direction. The rate of release of arginine from the enzyme-
arginine binary complex is 210 times faster than Vmax in the reverse
direction.
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