Kinetics competitive inhibition

A large number of amino acid transporters have been detected by isolating mutations which selectively inactivate one permease without altering enzyme activities involving the corresponding amino acid. Competitive inhibition, kinetics and regulatory behaviour have also been used as criteria to distinguish one transport system from another (see section 4.2). 

The most amplified amine, 22b, was resynthesized and assayed against the rate of HEWL lysis of Micrococcus lysodeikticus. Competitive inhibition kinetics was observed, with a recorded K value of 0.6 mM. The... 

However, an 18-residue segment of the autoinhibitory domain, composed of two short a-helical regions, was clearly visible in close contact with the catalytic domain, spanning the enzyme active site. This observation was consistent with previous studies showing competitive inhibition kinetics using a 25-residue peptide from the calcineurin A autoinhibitory domain (Parsons et al., 1994 Sagoo et al., 1996). 

If two different substrates bind simultaneously to the active site, then the standard Michaelis-Menten equations and competitive inhibition kinetics do not apply. Instead it is necessary to base the kinetic analyses on a more complex kinetic scheme. The scheme in Figure 6 is a simplified representation of a substrate and an effector binding to an enzyme, with the assumption that product release is fast. In Figure 6, S is the substrate and B is the effector molecule. Product can be formed from both the ES and ESB complexes. If the rates of product formation are slow relative to the binding equilibrium, we can consider each substrate independently (i.e., we do not include the formation of the effector metabolites from EB and ESB in the kinetic derivations). This results in the following relatively simple equation for the velocity ... 

The S1/S2 transition requires Cl but does not result in the release of a proton. From the interdependence of OH and Cl reported here, it is clear that H2O2 is formed when the concentration of OH is sufficient to displace Cl from the S2 state. Thus, we propose that Cl" inhibits the coordination of H2O (as the Lewis base OH") during the S1/S2 transition. This interaction between OH" and Cl" may explain the competitive inhibition kinetics of photosynthetic O2 evolution by OH" (14,17). 

Product inhibition studies are used to complement kinetic analyses and to distinguish between ordered and random Bi-Bi reactions. For example, in a random-order Bi-Bi reaction, each product will be a competitive inhibitor regardless of which substrate is designated the variable substrate. However, for a sequential mechanism (Figure 8-11, bottom), only product Q will give the pattern indicative of competitive inhibition when A is the variable substrate, while only product P will produce this pattern with B as the variable substrate. The... 

The cases of non-competitive inhibition and even more complex non-linear reaction kinetics will not be discussed further here. 

Two additional characteristics of the inhibition of mineral absorption by phenolic acids were observed. The inhibition of both P0 absorption (27) and K+ absorption (31, 32) was reversed when the phenolic acid was removed from the absorption solution. Harper Balke (32) found this reversibility to be dependent upon pH the lower the pH, the less the reversal. Also, kinetic plots of the inhibition of mineral absorption showed that the phenolic acids did not competitively inhibit either P0 (26, 28) or K+ (31) absorption. Rather, ferulic acid inhibited PO -absorption in a noncompetitive (26) or uncompetitive (28) manner and jr-hydroxybenzoic acid inhibited K+ absorption in an uncompetitive manner (31). 

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. 

Although kinetic evidence for prior equilibrium inclusion was not obtained, competitive inhibition by cyclohexanol and apparent substrate specificity once again provide strong support for this mechanism. Since the rate of the catalytic reaction is strictly proportional to the concentration of the ionized hydroxamate function (kinetic and spectrophotometric p/Cas are identical within experimental error and are equal to 8.5), the reaction probably proceeds by a nucleophilic mechanism to produce an acyl intermediate. Although acyl derivatives of N-alkylhydroxamic acids are exceptionally labile in aqueous solution, deacylation is nevertheless the ratedetermining step of the overall hydrolysis (Gruhn and Bender, 1969). 

Cyclodextrins as catalysts and enzyme models It has long been known that cyclodextrins may act as elementary models for the catalytic behaviour of enzymes (Breslow, 1971). These hosts, with the assistance of their hydroxyl functions, may exhibit guest specificity, competitive inhibition, and Michaelis-Menten-type kinetics. All these are characteristics of enzyme-catalyzed reactions. 

The kinetic parameters for the oxidation of a series of alcohols by ALD are shown in Table 4.1 (74). Methanol and ethylene glycol are toxic because of their oxidation products (formaldehyde and formic acid for methanol and a series of intermediates leading to oxalic acid for ethylene glycol), and the fact that their affinity for ALD is lower than that for ethanol can be used for the treatment of ingestion of these agents. Treatment of such patients with ethanol inhibits the oxidation of methanol and ethylene glycol (competitive inhibition) and shifts more of the clearance to renal clearance thus decreasing toxicity. ALD is also inhibited by 4-methylpyrazole. 

Another type of inhibitor combines with the enzyme at a site which is often different from the substrate-binding site and as a result will inhibit the formation of the product by the breakdown of the normal enzyme-substrate complex. Such non-competitive inhibition is not reversed by the addition of excess substrate and generally the inhibitor shows no structural similarity to the substrate. Kinetic studies reveal a reduced value for the maximum activity of the enzyme but an unaltered value for the Michaelis constant (Figure 8.7). There are many examples of non-competitive inhibitors, many of which are regarded as poisons because of the crucial role of the inhibited enzyme. Cyanide ions, for instance, inhibit any enzyme in which either an iron or copper ion is part of the active site or prosthetic group, e.g. cytochrome c oxidase (EC 1.9.3.1). 

Rate constants for the reaction of cytochrome f with inorganic oxidants are independent of pH when it is between 6.5-8.0 [150]. At pH < 6.5 protonation does have an effect, and with for example [Co(phen)3] there is a decrease and [Fe(CN)g] an increase in rate constants. From the kinetics cytochrome f shows no association with positively charged [Co(phen)j], and no competitive inhibition is observed with [Pt(NH3)6]" or [(NH3)5CoNH2Co(NH3)5] " [150]. It is possible therefore to study the effect of these reagents on the reaction of cytochrome f(II) with PCu(II), which... 

Thereafter, a reference text such as Enzyme Kinetics (Segel, 1993) should be consulted to determine whether or not the proposed mechanism has been described and characterized previously. For the example given, it would be found that the proposed mechanism corresponds to a system referred to as partial competitive inhibition, and an equation is provided which can be applied to the experimental data. If the data can be fitted successfully by applying the equation through nonlinear regression, the proposed mechanism would be supported further secondary graphing approaches to confirm the mechanism are also provided in texts such as Enzyme Kinetics, and values could be obtained for the various associated constants. If the data cannot be fitted successfully, the proposed reaction scheme should be revisited and altered appropriately, and the whole process repeated. 

In general, this first generation of abzymes obtained from TSAs behave like enzymes, present saturation kinetics, substrate specificity, a stereoselectivity and competitive inhibition phenomena. However the acceleration factors obtained, cat/ ncat> remain weak and are limited in theory by the ratio of the constant of... 

These equilibrium-binding relationships give rise to four different kinetic responses competitive inhibition, uncompetitive inhibition, non-competitive inhibition, mixed inhibition. Details of the kinetics of these types of inhibition and how dissociation constants for the reactions can be measured are provided in Appendix 3.6. 

Competition The rate of transport of one sugar is reduced by the transport of another sugar the kinetics are identical to that of competitive inhibition of hexokinase by the presence of other sugars. 

Figure 3. Kinetics of conq)etitivc inhibition of Clostridium thermohydrosuljur-icum strain 39E purified amylopuUulanase activity with mixed substrates. The solid lines A and C indicate the theoretical plots for competitive inhibition at amylose ccmcentrations of 0.6 and 2.4 mg/ml, respectively. Lines B and D are the theoretical plots for the absence of inhibition at the same respective amylose ccmcentrations. PuUulan was used at concentrations of 0.4, 0.8, 1.2, 1.6, 2.0, 2.4 mg/ml. For clarity, only two sets of data points were used in the above plot. ( ) and (A) are the practical data points obtained at 0.6 and 2.4 mg/ml amylose concentrations. All reaction mixtures contained 5% (v/v) dimethyl sulfoxide for solubility of amylose. [S] = [A] + [P], where S is the total substrate ccmcentration. A and P are the concentrations of amylose and pullulan, respectively. (Reproduced with permissiem from Ref. 13. Copyright 1990 Academic Press, Inc.)...

Carrier-mediated passage of a molecular entity across a membrane (or other barrier). Facilitated transport follows saturation kinetics ie, the rate of transport at elevated concentrations of the transportable substrate reaches a maximum that reflects the concentration of carriers/transporters. In this respect, the kinetics resemble the Michaelis-Menten behavior of enzyme-catalyzed reactions. Facilitated diffusion systems are often stereo-specific, and they are subject to competitive inhibition. Facilitated transport systems are also distinguished from active transport systems which work against a concentration barrier and require a source of free energy. Simple diffusion often occurs in parallel to facilitated diffusion, and one must correct facilitated transport for the basal rate. This is usually evident when a plot of transport rate versus substrate concentration reaches a limiting nonzero rate at saturating substrate While the term passive transport has been used synonymously with facilitated transport, others have suggested that this term may be confused with or mistaken for simple diffusion. See Membrane Transport Kinetics... 

For non-rapid-equilibrium cases (i.e., steady-state cases) the enzyme rate expression is much more complex, containing terms with [A] and with [I]. Depending on the relative magnitude of those terms in the initial rate expression, there may be nonlinearity in the standard double-reciprocal plot. In such cases, computer-based numerical analysis may be the only means for obtaining estimates of the magnitude of the kinetic parameters involving the partial inhibition. See Competitive Inhibition... 

Crude and three diethyl ether extracted, acetone treated, fractions were isolated from large-scale cultures of Gambierdiscus toxicus. Crude extracts at. 04 mg/ml inhibited the histamine contraction response in smooth muscle of the guinea pig ileum. Three semi-purified fractions at 5 ng/ml, effectively inhibited the guinea pig ileum preparation. Two of these fractions followed Michaelis-Menten kinetics for a competitive inhibition. The third fraction inhibited in a non-reversible manner. This study has established the presence of three lipid extracted toxins in toxicus, outlined a method for their assay in small quantities, and identified at least two of the effects of these toxic extracts in animals. 

The hydrogenation of nitrobenzene, nitrosobenzene and azobenzene has been studied singly and competitively. A kinetic isotope effect was observed with nitrobenzene but not with nitrosobenzene. Nitrosobenzene inhibits nitrobenzene hydrogenation in a competitive reaction, whereas azobenzene and nitrobenzene co-react but at lower rates. Taken together a more detailed mechanistic understanding has been obtained. 

Satisfaction of kinetic order. Carriers follow Michaelis-Menten-type saturation kinetics or first-order kinetics. Ion channels follow the type of respective structure—unimolecular transmembrane channels and bimolecular half-channels follow first- and second-order kinetics, respectively. The kinetic order of supramolecular channels depends on the assembly number. However, this principle can be applied only when the association constants are small. If the association becomes strong, the kinetic order decreases down to zero. Then the validity becomes dubious in view of the absolute criterion of the mechanism. Decreased activation energy compared to the carrier transport mechanism and competitive inhibition by added other cations stand as criteria. 

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