Enzyme kinetics competitive inhibition

Reversibly formed micelles have long been of interest as models for enzymes, since they provide an amphipathic environment attractive to many substrates. Substrate binding (non-covalent), saturation kinetics and competitive inhibition are kinetic factors common to both enzyme reaction mechanism analysis and micellar binding kinetics. 

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). 

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. 

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). 

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... 

While requiring the availability of competitive inhibitors for each of the substrates, Fromm s use of competitive inhibitors to distinguish multisubstrate enzyme kinetic pathways represents the most powerful initial rate method. See Alternative Substrate Inhibition... 

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... 

Also characteristic of enzymes that obey Michaelis-Menten kinetics is that suitable inhibitors can compete with the substrate for the enzyme active site, thus impeding the reaction. If the inhibitor binds reversibly to the enzyme active site, then the substrate can compete for the active site leading to competitive inhibition. To test for... 

Reversible Inhibition One common type of reversible inhibition is called competitive (Fig. 6-15a). A competitive inhibitor competes with the substrate for the active site of an enzyme. While the inhibitor (I) occupies the active site it prevents binding of the substrate to the enzyme. Many competitive inhibitors are compounds that resemble the substrate and combine with the enzyme to form an El complex, but without leading to catalysis. Even fleeting combinations of this type will reduce the efficiency of the enzyme. By taking into account the molecular geometry of inhibitors that resemble the substrate, we can reach conclusions about which parts of the normal substrate bind to the enzyme. Competitive inhibition can be analyzed quantitatively by steady-state kinetics. In the presence of a competitive inhibitor, the Michaelis-Menten equation (Eqn 6-9) becomes... 

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. 

Product inhibition (Section A,12) can also provide information about mechanisms. For example, if 1 / v is plotted against 1 / [A] in the presence and absence of the product Q, the product will be found to compete with A and to give a typical family of lines for competitive inhibition. On the other hand, a plot of 1 / v vs 1 / [B] in the presence and absence of Q will indicate noncompetitive inhibition if the binding of substrates is ordered (Eq. 9-43). In other words, only the A-Q pair of substrates are competitive. Product inhibition is also observed with enzymes having ping-pong kinetics (Eq. 9-47) as a result of formation of nonproductive complexes. 

Inhibition kinetics are included in the second category of assay applications. An earlier discussion outlined the kinetic differentiation between competitive and noncompetitive inhibition. The same experimental conditions that pertain to evaluation of Ku and Vmax hold for A) estimation. A constant level of inhibitor is added to each assay, but the substrate concentration is varied as for Ku determination. In summary, a study of enzyme kinetics is approached by measuring initial reaction velocities under conditions where only one factor (substrate, enzyme, cofactor) is varied and all others are held constant. 

These were differently affected by different procedures. For example, when the enzyme was activated at 55°, the increment in ki was slight, but k2 increased 3.5-fold. Similarly, in the presence of EDTA, fc, and k2 values decreased independently, suggesting that the sites for both activities were different. Center and Behai (5) found that with the P. mirabilis enzyme, cyclic 2, 3 -UMP competitively inhibited the hydrolysis of bis(p-nitrophenyl) phosphate. The Ki was 40 pAf very close to the Km for the cyclic nucleotide (Km, 75 yM) which indicated that the two compounds could serve as alternate substrates being hydrolyzed at the same active site. In contrast, 3 -AMP was a mixed inhibitor of cyclic 2, 3 -UMP and bis(p-nitrophenyl) phosphate hydrolysis. Adenosine was a mixed inhibitor of bis(p-nitrophenyl) phosphate hydrolysis but a competitive inhibitor of 3 -AMP hydrolysis. From such kinetic studies Center and Behai (5) suggested that two separate and adjacent sites A and B are involved in the hydrolysis of the diester and phos-phomonoester substrates. Site A serves as a binding site for hydrolysis of ribonucleoside 2, 3 -cyclic phosphates and together with site B catalyzes the hydrolysis of the diester bond. During this reaction 3 -... 

Crude estimates of the affinities of the enzyme for other compounds have been made by study of their capacity to inhibit hydrolysis of PPi. If the observed inhibition is assumed to be competitive, a simplified kinetic treatment yields the inhibition constants (Ki" values) listed in Table VI (54). The data indicate that several of the compounds whose hydrolysis is not catalyzed (Section III,D) are nevertheless bound weakly to the enzyme (e.g., ADP, methylene-bis-phosphonate). There is also very weak binding of Pi( the product of the enzymic reaction [Eq. 

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. 

Corey also pointed out that 16 reflects the transition-state of an enzyme-substrate complex. Its formation was later supported by the observation of Michaelis-Menten-type kinetics in dihydroxylation reactions and in competitive inhibition studies [37], This kinetic behavior was held responsible for the non-linearity in the Eyring diagrams, which would otherwise be inconsistent with a concerted mechanism. Contrary, Sharpless stated that the observed Michaelis-Menten behavior in the catalytic AD would result from a step other than osmylation. Kinetic studies on the stoichiometric AD of styrene under conditions that replicate the organic phase of the catalytic AD had revealed that the rate expression was clearly first-order in substrate over a wide range of concentrations [38],... 

In Table II are shown the results from kinetic studies with commercially available gastric and pancreatic enzymes. Trypsin was strongly inhibited, at least at a low concentration of casein as substrate. The hydrolysis of benzoyl arginine ethyl ester (BAEE) by trypsin was non-competitively inhibited, giving a 30% reduction of Vmax at 0.5 mg/ml of the LMW fraction. Carboxypepti-dase A, and to a lesser extent carboxypeptidase B, were non-competitively inhibited as well. Pepsin and chymotrypsin were not affected by the conditions used in these assays. 

This mechanism is important for compounds that lack sufficient lipid solubility to move rapidly across the membrane by simple diffusion. A membrane-associated protein is usually involved, specificity, competitive inhibition, and the saturation phenomenon and their kinetics are best described by Michaelis-Menton enzyme kinetic models. Membrane penetration by this mechanism is more rapid than simple diffusion and, in the case of active transport, may proceed beyond the point where concentrations are equal on both... 

The primary considerations in studies of inhibition mechanisms are reversibility and selectivity. The inhibition kinetics of reversible inhibition give considerable insight into the reaction mechanisms of enzymes and, for that reason, have been well studied. In general, reversible inhibition involves no covalent binding, occurs rapidly, and can be reversed by dialysis or, more rapidly, by dilution. Reversible inhibition is usually divided into competitive inhibition, uncompetitive inhibition, and noncompetitive inhibition. Because these types are not rigidly separated, many intermediate classes have been described. 

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). 

For a detailed review of simple to complex enzyme kinetics, a book by Segel (21) is recommended. Most P450 oxidations show hyperbolic saturation kinetics and competitive inhibition between substrates. Therefore, both Km values and drug interactions can be predicted from inhibition studies. Competitive inhibition suggests that the enzymes have a single binding site and only one substrate can bind at any one time. For the inhibition of substrate A by substrate B to be competitive, the following must be observed ... 

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