Dead-end inhibition

The example of methotrexate points out that the inhibition modality of dead end inhibitors, with respect to a specific substrate, will depend on the reaction mechanism of the target enzyme. Thus a complete understanding of inhibition mechanism requires an understanding of the underlying reaction mechanism of the target enzyme. A comprehensive discussion of these issues has been provided by Segel (1975). Table 3.6 summarizes the pattern of dead-end inhibition observed for competitive inhibitors of one substrate in the common bisubstrate reaction mecha-... 

Table 3.6 Pattern of dead-end inhibition observed for bisubstrate reactions...

Cleland presented more complex examples of multiple dead-end inhibition in which the kinetic expression for the slope or intercept is a function containing polynomials of the inhibitor concentration in both the denominator and numerator. For example, if the slope is equal to a function having the form (a + b[ ] + c[I] )/(d -f c[I]) in which a, b, c, d, and e are constants or collections of constants, then the slope is said to be a 2/1 function (the numbers representing the highest power of [I] in the numerator and denominator, respectively). The nonlinearity of the slope replot, in this case, is dependent on the relative magnitudes of the constants in the expression. 

RANDOM SCISSION KINETICS MULTIPLE DEAD-END INHIBITION MULTIPLE-TURNOVER CONDITIONS MULTIPLICATIVE MODEL MULTIPLICITY... 

In this model the unimolecular constants are relative to the turnover number and the bimolecular constants are chosen to yield equilibrium constants in units of millimolar. The model is primarily based on dead-end inhibition by CrATP, the Michaelis constant for ATP in the ATPase reaction, the isotope partitioning experiments of Rose et al. (65), and various binding and kinetic constants found in the literature. The final model was based on a computer simulation study attempting to discover what combination of rate constants would lit the isotope partition data and the observed kinetic and binding constants. 

Hexokinase does not yield parallel reciprocal plots, so the Ping Pong mechanism can be discarded. However, initial velocity studies alone will noi discriminate between the rapid equilibrium random and steady-state ordered mechanisms. Both yield ihe same velocity equation and families of intersecting reciprocal plots. Other diagnostic procedures must be used (e.g., product inhibition, dead-end inhibition, equilibrium substrate binding, and isotope exchange studies). These procedures are described in detail in the author s Enzyme Kinetics behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems, Wiley-Interscience (1975),... 

Three mies can be formulated to predict product or dead-end inhibition patterns in such mechanisms. (1) When an inhibitor occupies the same portion of an active site as the variable substrate, the inhibition is competitive. (2) When an inhibitor combines in a different portion of the same active site as the variable substrate, the inhibition is noncompetitive. Thus, with pyruvate carboxylase, MgADP and P are both noncompetitive inhibitors versus bicarbonate, although in accordance with Rule 1 they are competitive versus MgATP. (3) When an... 

Although the basis of product inhibition is somewhat different from that of dead-end inhibition, the end results may nevertheless be categorised in the same way, depending upon whether the inhibitor affects the slopes, the intereepts or both in plots of e/v against 1/[S]. Thus the terms competitive, non-competitive, uncompetitive and mixed still apply. If we consider Eqn. 17, a slope effect will be seen, with A as the varied substrate, if the inhibitor affects the term, the both ... 

DEAD-END INHIBITION IN STEADY-STATE BISUBSTRATE SYSTEMS... 

Figure 5. Dead-end inhibition in a Ping Pong Bi Bi system, when an inhibitor binds to the free en me, producing an El complex. Graphical presentation of Eqs. (5.28) and (5.29).

Dead-end Inhibition in a Rapid Equilibrium Ordered Bisubstrate System... 

In Section 5.4, a case of a dead-end inhibition in a Rapid Equilibrium Ordered bisubstrate system was described. One can compare this system with the following example. 

Dead-end Inhibition in a Steady-State Ordered Bi Bi System In this system, a frequent case is when a dead-end inhibitor combines with EA... 

Figure 9 shows the graphical presentation of Eq. (1134), a plot of i/Uo versus 1/5, in the presence of increasing concentrations of P. The double reciprocal plot has an unusual appearance. Thus, the mixed product and dead-end inhibition by P can be distinguished from the normal product inhibition by P from the fact that the family of straight lines does not intersect at a common point. 

Figure 9. Mixed product and dead-end inhibition in an Ordered Bi Bi system. P reacts with EA as well as with EQ (Scheme (11.31)). The plot of i/un versus l/B is slope-parabolic, intercept-linear.

The situation shown in Eq. (11.39) is analogous to the dead-end inhibition by a nonreacting inhibitor, described in Chapter 6 (Section 6.5.1). The rate equation is derived in the usual manner both B and P terms in the distribution equation are multiplied by (i+P/fTi), to obtain... 

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