Double reciprocal plot sequential mechanism

A procedure used to assist in identifying sequential mechanisms when the double-reciprocal plots exhibit parallel lines ". In some cases, bireactant mechanism can have various collections of rate constants that result in so-called parallel line kinetics, even though the mechanism is not ping pong. However, if the concentrations of A and B are kept in constant ratio with respect to each other, a sequential mechanism in a 1/v v. 1/[A] plot would be nonlinear (since in the denominator the last term of the double-reciprocal form of the rate expression contains [A] for example, for the steady-state ordered Bi Bi reaction scheme in which [B] = a[A], the double-reciprocal rate expression becomes 1/v =... 

Figure 5.10 Double-reciprocal plots of two-substrate reactions at three different concentrations of B. (A) Sequential reaction mechanism (B) ping-pong mechanism.

Figure. 6.13. Double reciprocal plot for sequential mechanism.

The general property of all above mechanisms is their adherence to the Michaelis-Menten kinetics. In the absence of products, the double reciprocal plots for aU bisubstrate mechanisms represent a family of straight lines with a common intersection point, if one substrate is varied while the other substrate is held at different fixed concentrations. Similarly, the double reciprocal plots for all trisubstrate mechanisms represent a family of straight lines with a common intersection point, if one substrate is varied while the second substrate is held at different fixed concentrations, and the third substrate is held at a fixed concentration. This, however, is tme only if each substrate adds just once if one of them adds twice in sequential fashion, the reciprocal plots will be parabolic. 

Figure 7 Cleland plots of the different mechanisms in multisubstrate enzyme-catalyzed reactions (A) sequential mechanism, ordered (left) and random (right) (B) ping-pong mechanism. The double reciprocal plots identify either mechanism. Ordered and random show identical data display. 1,2, 3.5 denote increasing concentrations of the second substrate.

Fig. 1. Effect of agmatine and NAD on the activity of NAD arginine ADP-ribosyltransferase plotted with NAD as the variable substrate at different fixed concentrations of agmatine. Assays contained 50 mM potassium phosphate (pH 7.0), 1 mg ml ovalbumin, and the following concentrations of agmatine o 0.5 mAf 1 mAf 2 mM O 3 mAf. Reactions were performed at 30 C and initiated with erythrocyte transferase A (9.1 ng/assay). Inset Double reciprocal plot of the activity of transferase A in the absence of acceptor. Solid lines are the theoretical fit of the data to the random rapid equilibrium sequential mechanism described in the text...

The kinetic mechanisms of PP-ribose-P synthetase partially purified from human erythrocytes has been studied. Double reciprocal plots of initial velocity studies with variable MgATP cencen-trations and fixed ribose-5-P are intersecting and suggest a sequential kinetic mechanism, (Figure 2). Product inhibition studies... 

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