Velocity inhibitors affecting

An inhibitor that binds exclusively to the ES complex, or a subsequent species, with little or no affinity for the free enzyme is referred to as uncompetitive. Inhibitors of this modality require the prior formation of the ES complex for binding and inhibition. Hence these inhibitors affect the steps in catalysis subsequent to initial substrate binding that is, they affect the ES —> ES1 step. One might then expect that these inhibitors would exclusively affect the apparent value of Vm and not influence the value of KM. This, however, is incorrect. Recall, as illustrated in Figure 3.1, that the formation of the ESI ternary complex represents a thermodynamic cycle between the ES, El, and ESI states. Hence the augmentation of the affinity of an uncompetitive inhibitor that accompanies ES complex formation must be balanced by an equal augmentation of substrate affinity for the El complex. The result of this is that the apparent values of both Vmax and Ku decrease with increasing concentrations of an uncompetitive inhibitor (Table 3.3). The velocity equation for uncompetitive inhibition is as follows ... 

Figure 6.2 Effect of preincubation time with inhibitor on the steady state velocity of an enzymatic reaction for a very slow binding inhibitor. (A) Preincubation time dependence of velocity in the presence of a slow binding inhibitor that conforms to the single-step binding mechanism of scheme B of Figure 6.3. (B) Preincubation time dependence of velocity in the presence of a slow binding inhibitor that conforms to the two-step binding mechanism of scheme C of Figure 6.3. Note that in panel B both the initial velocity (y-intercept values) and steady state velocity are affected by the presence of inhibitor in a concentration-dependent fashion.

At low concentrations of substrate ([S] < Km), the enzyme is predominantly in the E form. The competitive inhibitor can combine with E, so the presense of the inhibitor decreases the velocity when the substrate concentration is low. At low substrate concentration ([S] < Km), the velocity is just Vmay IKm. Since the inhibitor decreases the velocity and the velocity at low substrate concentration is proportional to Vmax/Km, the presence of the inhibitor affects the slopes of the Lineweaver-Burk plots the slope is just the reciprocal of Vmax/Km. Increasing the inhibitor concentration causes Km/Vmax to increase. The characteristic pattern of competitive inhibition can then be rationalized if you simply remember that a competitive inhibitor combines only with E. 

The most important factors affecting performance are operating temperature, surface velocity, contaminant concentration and composition, catalyst properties, and the presence or absence of poisons or inhibitors. 

An inhibitor that binds exclusively to the free enzyme (i.e., for which a = °°) is said to be competitive because the binding of the inhibitor and the substrate to the enzyme are mutually exclusive hence these inhibitors compete with the substrate for the pool of free enzyme molecules. Referring back to the relationships between the steady state kinetic constants and the steps in catalysis (Figure 2.8), one would expect inhibitors that conform to this mechanism to affect the apparent value of KM (which relates to formation of the enzyme-substrate complex) and VmJKM, but not the value of Vmax (which relates to the chemical steps subsequent to ES complex formation). The presence of a competitive inhibitor thus influences the steady state velocity equation as described by Equation (3.1) ... 

An inhibitor can have different effects on the velocity when the substrate concentration is varied. If the inhibitor and substrate compete for the same form of the enzyme, the inhibition is COMPETITIVE. If not, the inhibition is either NONCOMPETITIVE or UNCOMPETITIVE depending on whether or not the inhibitor can affect the velocity at low substrate concentrations. 

At very low substrate concentration ([S] approaches zero), the enzyme is mostly present as E. Since an uncompetitive inhibitor does not combine with E, the inhibitor has no effect on the velocity and no effect on Vmsa/Km (the slope of the double-reciprocal plot). In this case, termed uncompetitive, the slopes of the double-reciprocal plots are independent of inhibitor concentration and only the intercepts are affected. A series of parallel lines results when different inhibitor concentrations are used. This type of inhibition is often observed for enzymes that catalyze the reaction between two substrates. Often an inhibitor that is competitive against one of the substrates is found to give uncompetitive inhibition when the other substrate is varied. The inhibitor does combine at the active site but does not prevent the binding of one of the substrates (and vice versa). 

Substrates can affect the conformation of the other active sites. So can other molecules. Effector molecules other than the substrate can bind to specific effector sites (different from the substrate-binding site) and shift the original T-R equilibrium (see Fig. 8-9). An effector that binds preferentially to the T state decreases the already low concentration of the R state and makes it even more difficult for the substrate to bind. These effectors decrease the velocity of the overall reaction and are referred to as allosteric inhibitors. An example is the effect of ATP or citrate on the activity of phosphofructokinase. Effectors that bind specif-... 

Reversible inhibition occurs rapidly in a system which is near its equilibrium point and its extent is dependent on the concentration of enzyme, inhibitor and substrate. It remains constant over the period when the initial reaction velocity studies are performed. In contrast, irreversible inhibition may increase with time. In simple single-substrate enzyme-catalysed reactions there are three main types of inhibition patterns involving reactions following the Michaelis-Menten equation competitive, uncompetitive and non-competitive inhibition. Competitive inhibition occurs when the inhibitor directly competes with the substrate in forming the enzyme complex. Uncompetitive inhibition involves the interaction of the inhibitor with only the enzyme-substrate complex, while non-competitive inhibition occurs when the inhibitor binds to either the enzyme or the enzyme-substrate complex without affecting the binding of the substrate. The kinetic modifications of the Michaelis-Menten equation associated with the various types of inhibition are shown below. The derivation of these equations is shown in Appendix S.S. 

A classic example of competitive inhibition is the inhibition of succinate dehydrogenase by malonate, a structural analogue of succinate. Competitive inhibitors are usually structural analogues of the substrate, the molecule with which they are competing. They bind to the active site but either do not have a structure that is conducive to enzymatic modification or do not induce the proper orientation of catalytic amino acyl residues required to affect catalysis. Consequently, they displace the substrate from the active site and thereby depress the velocity of the reaction. Increasing [S] will displace the inhibitor. 

From Equation 17.21 it is clear that noncompetitive inhibitors have an effect only on max> decreasing it by a factor of (1 + [Il/ifi), consequently giving the impression of reducing the total amount of enzyme present. As with an uncompetitive inhibitor, a portion of the enzyme will always be bound in the nonproductive enzyme-substrate-inhibitor complex E e S I, causing a decrease in maximum velocity, even at infinite substrate concentrations. However, because noncompetitive inhibitors do not affect substrate binding, the Km value of the substrate remains unchanged. Linear plots for noncompetitiveinhibition are shown in Fig. 17.9. 

In order to study how L-phenylalanine affects the rate of decomposition of ES to products, ks, the first-order velocity constant for the decomposition, was determined with and without the inhibitor, using 0.05 M carbonate-bicarbonate buffer at pH 9.2. Km, necessary for the calculation of fcs, was obtained according to the method of Veibel and Lillelund as... 

In noncompetitive inhibition (Figure 8.38), the inhibitor can combine with either the enzyme or the enzyme-substrate complex. In pure noncompetitive inhibition, the values of the dissociation constants of the inhibitor and enzyme and of the inhibitor and enzyme—substrate complex are equal (Section 8.5.1). The value of is decreased to a new value called V( i. and so the intercept on the vertical axis is increased. The new slope, which is equal to Km/ V( i. is larger by the same factor. In contrast with Vjjxix. is not affected by pure noncompetitive inhibition. The maximal velocity in the presence of a pure noncompetitive inhibitor, V ax. is given by... 

Competitive inhibitors decrease the velocity of an enzymatic reaction by increasing the amount of substrate required to saturate the enzyme therefore, they increase the apparent Km but do not affect Vmax. A Lineweaver-Burk plot of a competitively inhibited enzyme reaction has an increased slope, but its intercept is unchanged. 

The uncompetitive inhibitor binds to the enzyme-substrate complex, but not to the free enzyme. Both and Vmax are affected, but the ratio KM/Vmax remains constant. The reaction velocity obeys ... 

P, M and L are the pol3mier, metal and lubricating medium P, 1/, Cl are the frictional factors, i.e. pressure, velocity and the presence of Cl in the friction zone TCRP are the tribochemical reaction products. The latter can fulfill the function of wear inhibitors (WI) during physical-chemical interactions with the inhibitor on the metal friction surface or form neutral wear products (NWP) affecting neither corrosion nor friction. The task is how to transform these products into useful ones during friction. 

It is apparent from Scheme 4.3 that the inhibitor cannot only compete with substrate for binding to the enzyme but bind to an enzyme molecule that subsequently binds a substrate molecule also or to an enzyme-substrate complex to affect catalytic turnover. These multiple binding mechanisms help explain the effects of mixed inhibition on both Vmax and K. The effects of mixed inhibition on the velocity of the reaction can be described by the following mixed inhibition equation (Eq. 4.15) ... 

Since these experiments were not carried out under ideally defined flow conditions the dependence of corrosion rate on flow rate will be discussed only in a qualitative manner. Under laminar flow conditions and mass transfer control one would have expected the corrosion rate to increase with the square root of the velocity while under turbulent conditions proportionality would prevail. However, in Fig.15 one finds that the corrosion rate varies approximately with the 0.2 to 0.3 power of the flow rate. It appears therefore that the observed dependence on the flow rate does not obey conventional mass transfer theory. A flow effect might be expected in uninhibited hydrochloric acid because hydrogen bubbles, formed on the surface of the metal, are faster and more easily removed at higher flow rates. While this argument could be applied in discussing Fig.15, we find in Fig.16 that the flow effect at similar corrosion rates is much less pronounced under deaerated conditions. We therefore have to conclude that the observed flow effect is not mechanical and cannot be related to pure mass transfer control either. In Fig.17, the flow dependence of the corrosion rate is shown for 2-butyne-l,4-diol in deaerated UN hydrochloric acid. Note that the corrosion rate appears to be noticeably affected only at the higher flow rates. Finally, in Fig.18, we observe that increased flow rate can either increase or decrease the corrosion rate in the presence of an inhibitor. This effect was observed reproducibly only in 6N hydrochloric acid with 2-butyne-l,U-diol under deaerated conditions for 0.2% and 0.1% inhibitor concentration. This behavior indicates that the corrosion rate is controlled by the superposition of two partial reaction rates each of which is mass transfer dependent to a certain extent. In terms of the model delineated in Table 6, it is suggested that the three-dimensional polymeric layer made up by inhibitor molecules is in fact a three-dimensional chelate made up of iron ions and inhibitor molecules. The corrosion rate is then... 

An example of corrosion in a 22-inch (56 cm) flow line for oil and gas at a field in the North Sea is shown in Figure 7.44. During a service period of fln-ee years attacks as deep as 6.5 mm have developed. The temperature and the pressure (at least in periods) have been 75-80°C and 60-80 bar. Inhibitor treatment was carried out during the last half year before the actual pipe section was removed. The flow velocity has been 4—5 m/s most of the time. The flow conditions have not been extreme, but the shape and position of the attacks show that they are flow-affected. It is assumed that corrosion can be prevented if the temperature on the inner pipe wall is kept above the dewpoint of the water vapour in the gas, which may possibly be achieved by arrangements for reduction of the heat loss to the surroundings or change of the internal pressure and temperature. An alternative measure is to reduce the flow rate combined with appropriate addition of inhibitor. 

To see how velocity equations are affected by a dead-end inhibitor, let us illustrate the above rule with several examples. 

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