Enzymatic kinetics inhibition

We will consider in this chapter the general processes by which enzymes achieve enhancement of reaction rates, basic chemical and enzymatic kinetics and inhibition, the roles of cofactors and coenzymes, the effects of environmental factors, the regulation of enzyme activity, and some clinical applications of enzymology. 

To fully establish whether arogenate (41), phenylpyruvate (39), or both were pathway intermediates to Phe (1) in vascular plants, it was essential to unambiguously identify all enzymatic processes (and encoding genes) needed for conversion of prephenate (38) into Phe (1) both in vivo and in vitro. Moreover, it was also essential to obtain rigorous enzymatic kinetic data using highly purified recombinant enzymes in vitro for all potential substrates, in order to compare and contrast relative efficacies/feedback inhibition properties and so forth. 

ESI (LC-MS) Kinetic study of enzymatic reaction, inhibition Simithy eta/. [337]... 

The products of the reaction they catalyze may inhibit many enzymes through Michaelis-Menten kinetic retroaction. Protons, which are involved as products or reactants in a number of cases, may also influence the enzymatic kinetics. The course of the reaction may therefore be altered by the attending production or depletion of protons. It is thus interesting to examine whether these phenomena may be revealed by the effect they might have on the electrochemical responses of immobilized enzyme films under appropriate conditions [92]. A first clue of the existence of such inhibition effects is the observation of hysteresis behaviors of the type shown in Fig. 18(a) where data obtained with 10 glucose oxidase monolayers with ferrocene methanol as cosubstrate have been taken as example. In the absence of inhibition, the forward and reverse traces should be exactly superimposed. Hysteresis increases to the point of making a peak appear on the forward trace as the scan rate decreases and as the concentration of the buffer decreases, as illustrated in Fig. 18c, c , c , c by comparison with Fig. 18(a). 

FIGURE 57.4 Kinetic inhibition 3D. Representation of the inhibition kinetics of soluble peripheral nerve PV esterase activity PVases by DFP. Inhibitory surface obtained by fitting the 3D model equation to the data corresponding to DFP inhibition. The surface reflects the result of the best model according to the F test. It corresponds to a model with two sensitive enzymatic components plus other resistant components. 

The impact of inhibition on the enzymatic kinetics is lower in the presence of external diffusion resistance, since mass transfer limitations make kinetic control less prominent. 

An enzymatic assay can also be used for detecting anatoxin-a(s). " This toxin inhibits acetylcholinesterase, which can be measured by a colorimetric reaction, i.e. reaction of the acetyl group, liberated enzymatically from acetylcholine, with dithiobisnitrobenzoic acid. The assay is performed in microtitre plates, and the presence of toxin detected by a reduction in absorbance at 410 nm when read in a plate reader in kinetic mode over a 5 minute period. The assay is not specific for anatoxin-a(s) since it responds to other acetylcholinesterase inhibitors, e.g. organophosphoriis pesticides, and would need to be followed by confirmatory tests for the cyanobacterial toxin. 

If the velocity of an enzymatic reaction is decreased or inhibited, the kinetics of the reaction obviously have been perturbed. Systematic perturbations are a basic tool of experimental scientists much can be learned about the normal workings of any system by inducing changes in it and then observing the effects of the change. The study of enzyme inhibition has contributed significantly to our understanding of enzymes. 

If the inhibitor combines irreversibly with the enzyme—for example, by covalent attachment—the kinetic pattern seen is like that of noncompetitive inhibition, because the net effect is a loss of active enzyme. Usually, this type of inhibition can be distinguished from the noncompetitive, reversible inhibition case since the reaction of I with E (and/or ES) is not instantaneous. Instead, there is a time-dependent decrease in enzymatic activity as E + I El proceeds, and the rate of this inactivation can be followed. Also, unlike reversible inhibitions, dilution or dialysis of the enzyme inhibitor solution does not dissociate the El complex and restore enzyme activity. 

The pH dependence of HIV-1 protease has been assessed by measuring the apparent inhibition constant for a synthetic substrate analog (b). The data are consistent with the catalytic involvement of ionizable groups with pK values of 3.3 and 5.3. Maximal enzymatic activity occurs in the pH range between these two values. On the basis of the accumulated kinetic and structural data on HIV-1 protease, these pK values have been ascribed to the... 

FIGURE 12.1 Effects of substrate (reactant) concentration on the rate of enzymatic reactions (a) simple Michaelis-Menten kinetics (b) substrate inhibition (c) substrate activation. 

The kinetic equation used here is an enzymatic Michaelis-Menten form with product inhibition... 

More recent detailed kinetic investigations have revealed that actinonin is a time-dependent, essentially irreversible, inhibitor of PDF enzymatic activity [72]. This study demonstrated that the kinetics of inhibition of... 

If more than one substrate participates in an enzymatic reaction, the kinetic effects of an inhibitor can be quite complex. In this case, rules formulated by Cleland (36) are useful in gaining a qualitative picture of the inhibition patterns to be expected of a given mechanism. 

A final source of evidence for the formation of inclusion complexes in solution has been derived from kinetic measurements. Rate accelerations imposed by the cycloamyloses are competitively inhibited by the addition of small amounts of inert reagents such as cyclohexanol (VanEtten et al., 1967a). Competitive inhibition, a phenomenon frequently observed in enzymatic catalyses, requires a discrete site for which the substrate and the inhibitor can compete. The only discrete site associated with the cycloamyloses is their cavity. 

The enzymatic activity of the L-19 IVS ribozyme results from a cycle of transesterification reactions mechanistically similar to self-splicing. Each ribozyme molecule can process about 100 substrate molecules per hour and is not altered in the reaction therefore the intron acts as a catalyst. It follows Michaelis-Menten kinetics, is specific for RNA oligonucleotide substrates, and can be competitively inhibited. The kcat/Km (specificity constant) is 10s m- 1 s lower than that of many enzymes, but the ribozyme accelerates hydrolysis by a factor of 1010 relative to the uncatalyzed reaction. It makes use of substrate orientation, covalent catalysis, and metalion catalysis—strategies used by protein enzymes. 

Kinetic analysis was used to characterize enzyme-catalyzed reactions even before enzymes had been isolated in pure form. As a rule, kinetic measurements are made on purified enzymes in vitro. But the properties so determined must be referred back to the situation in vivo to ensure they are physiologically relevant. This is important because the rate of an enzymatic reaction can depend strongly on the concentrations of the substrates and products, and also on temperature, pH, and the concentrations of other molecules that activate or inhibit the enzyme. Kinetic analysis of such effects is indispensable to a comprehensive picture of an enzyme. 

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