Enzymatic competitive inhibitor

It is revealing to compare the equation for the uninhibited case. Equation (14.23) (the Michaelis-Menten equation) with Equation (14.43) for the rate of the enzymatic reaction in the presence of a fixed concentration of the competitive inhibitor, [I]... 

Each of these compounds, 53-56, was shown to be a very effective competitive inhibitor of the enzyme with respect to the fructose 1,6-diphosphate, whereas several other analogs, including acyclic structures, had no effect. These and other results suggest that the furanose form of the sugar diphosphate is the active form in the enzymatic reaction (105). More recent studies using rapid quenching techniques and C-nmr measurements have confirmed this hypothesis and indicate that the enzyme uses the a anomer 52 much more rapidly than the 3 anomer 50 and probably uses the a anomer exclusively (106). 

In Chapter 3 we saw that inhibitors of different modalities respond differently to the concentration of substrate used in an enzymatic reaction. Recall that the apparent affinity of the free enzyme for substrate was diminished in the presence of a competitive inhibitor, and vice versa, the apparent affinity of a competitive inhibitor could be abrogated at high substrate concentrations. On the other hand, the appar-... 

Enzymatic reactions can be impeded by the addition of exogenous molecules. This is how drugs are used to control biochemical reactions, and most drugs are used for inhibitory functions. Drugs may function as competitive inhibitors or as noncompetitive inhibitors. Competitive inhibitors compete with the substrates for binding to the active sites, whereas noncompetitive inhibitors bind to another location (allosteric site) but affect the active site and its consequential interactions with the substrates. Some drugs used as enzyme inhibitors are the following ... 

Hyperbolic curve fits to control enzymatic data and to data obtained in the presence of a competitive inhibitor. Curve fitting to the Michaelis-Menten equation results in two different values for Km- However, Km does not, in actuality, change, and the value in the presence of inhibitor (15 uiM) is an apparent value. Fitting with the correct equation, that for turnover in the presence of a competitive inhibitor ( Eq. 5), results in plots identical in appearance to those obtained with the Michaelis-Menten equation. However, nonlinear regression now reveals that Km remains constant at 5 ulM and that [l]/Ki = 2.5 with knowledge of [/], calculation of K is straightforward... 

The fact that ATP and CTP bind to the same site follows from the observation that adding ATP to the inhibited enzyme by CTP reduces or reverses the inhibition, presumably because ATP competes with CTP for the same site. The fact that CTP binds to an allosteric site (i.e., it is not a competitive inhibitor) follows from the so-called desensitization effect. Addition of mercurials [e.g., p-mercuribenzoate (PMB)] reduces or eliminates the inhibition by CTP. However, it has no effect on the enzymatic activity of ATCase, presumably because the mercurials affect the regulatory subunits but not the catalytic site. As for the mechanism of cooperativity (both positive and negative), it is known that CTP does induce changes in the quaternary structure of the enzyme. 

Although less explored, drug discovery efforts have also been directed to modulate IGF-IR kinase activity by compounds that do not necessarily interact with the ATP-binding cleft. Initial attempts to inhibit IGF-IR enzymatic activity with non-ATP competitive inhibitors resulted in the identification of several tyrphostin-type compounds (e.g., compound 4, Fig. 2) that showed weak activity in blocking IGF-IR autophosphorylation (IC50 7-13 p.M), but... 

General aspects of enzymatic reactions cateuLyzed by kinases are briefly mentioned. Many alternate substrates, competitive inhibitors and affinity labels based either on the structure of ATP or on the structure of the non-ATP kinase substrates are described. Several examples are presented that should be of particular interest to the medicinal chemist. Finally, the design of an affinity label for creatine kinase is reviewed as an example of how such information can be used in the search for agents directed at an enzyme s active site. 

Kinetic investigations were performed using the tetrasaccharide (44i), its higher homologs of DP 5 and DP 6, and 1,3 l,4-j3-D-glucanase isolated from Bacillus licheniformis [42 b]. As expected, all these compounds were resistant to enzymatic cleavage and have been shown to act as competitive inhibitors (Ki in mmol/1 range). 

Impressed by the specificity of enzymatic action, biochemists early adopted a "lock-and-key" theory which stated that for a reaction to occur the substrate must fit into an active site precisely. Modem experiments have amply verified the idea. A vast amount of kinetic data on families of substrates and related competitive inhibitors support the idea and numerous X-ray structures of enzymes with bound inhibitors or with very slow substrates have given visual evidence of the reality of the lock-and-key concept. Directed mutation of genes of many enzymes of known three-dimensional structure has provided additional proof. 

Various 3-halopyruvate derivatives have been reported to be decarboxylated and simultaneously dehalogenated, yielding acetate, carbon dioxide and a halogenide ion as the only reaction products [133,158], A similar reaction may occur upon enzymatic decarboxylation of 3-hydroxypyruvate, which is an alternative substrate and a strong competitive inhibitor of PDC [159],... 

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. 

As previously mentioned, many of the enzymes involved in xenobiotic metabolism are inducible. Inducibility allows for more enzymatic activity, thereby ensuring an adequate detoxication response however, it also provides a mechanism whereby an activation pathway may be increased. This occurs in the example given earlier of the combined effects of ethanol and acetaminophen. When CYP2E1 is induced by ethanol prior to administration of acetaminophen, subsequent activation of acetaminophen to NAPQI is prevalent however, without induction by ethanol, CYP2E1 is not the predominant enzyme for metabolizing acetaminophen, and detoxication is favored. Interestingly, simultaneous administration decreases the toxicity of acetaminophen because both are substrates for 2E1 ethanol acts as a competitive inhibitor, thereby blocking the activation of acetaminophen. 

Nucleotides in the form of metal ion complexes are involved in a variety of enzymatic reactions either as substrates or as cofactors. These may also be viewed as monomers of DNA and RNA. Lanthanide complexes of nucleotides have been extensively studied because (i) the conformation of nucleotides in solution can be elucidated from lanthanide induced NMR chemical shifts and line-broadenings [40] and (ii) lanthanide nucleotide complexes may act as competitive inhibitors in enzymatic reactions [88] and hence can be used as paramagnetic probes in the mapping of their binding site on the enzyme [89]. 

Figure 2 Stoichiometry of the enzymatic mechanism of formation of NO, and the structure of a competitive inhibitor, N -monomethyl-L-arginine (NMMA). NO is synthesized by all NOS s by a similar mechanism, involving the NADPH-dependent mixed-function oxidation of a guanidino nitrogen of the amino acid L-arginine (L-arg) to produce L-citrulline (L-cit) and -NO. The nonintegral stoichiometries are explained in the text. NMMA inhibits NOS as a competitive inhibitor...

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