Substrate analogs, competitive inhibition

Competitive inhibitors are inhibitors which have an effect on the but not on the V of an enzyme-catalysed reaction. The V is unchanged because the number of functional active sites is not altered but a greater substrate concentration is required to achieve the maximum utilization of the sites. Consequently, the for the substrate increases. Competitive inhibition may be overcome by the addition of more substrate to the enzyme reaction mixture. Competitive inhibitors often bear a structural similarity to the substrate and compete with the substrate for the active sites of the enzyme, i.e. they are isosteric. However, competitive inhibitors are not necessarily structurally analogous to the substrate, e.g. salicylate inhibition of 3-phospho-glycerate kinase, and may bind to a site distinct from the active site, e.g. L-isoleucine inhibition of threonine deaminase from Escherichia coli. The classical example of competitive inhibition is the action of malonate on succinate dehydrogenase (Figure 6.9) which advanced the elucidation of the... 

Probably all adenylyl cyclases are inhibited competitively by substrate analogs, which bind at the site and to the enzyme configuration with which cation-ATP binds (cf Fig. 4). One of the best competitive inhibitors is (3-L-2, 3 -dideoxy adenosine-5 -triphosphate ( 3-L-2, 3 -dd-5 -ATP Table 4) [4], which allowed the identification of the two metal sites within the catalytic active site (cf Fig. 4) [3]. This ligand has also been labeled with 32P in the (3-phosphate and is a useful ligand for reversible, binding displacement assays of adenylyl cyclases [4]. The two inhibitors, 2, 5 -dd-3 -ATP and 3-L-2, 3 -dd-5 -ATP, are comparably potent... 

Substrate analogs (2) have properties similar to those of one of the substrates of the target enzyme. They are bound by the enzyme, but cannot be converted further and therefore reversibly block some of the enzyme molecules present. A higher substrate concentration is therefore needed to achieve a halfmaximum rate the Michaelis constant increases (B). High concentrations of the substrate displace the inhibitor again. The maximum rate V ax is therefore not influenced by this type of inhibition. Because the substrate and the inhibitor compete with one another for the same binding site on the enzyme, this type of inhibition is referred to as competitive. Analogs of the transition state (3) usually also act competitively. 

Competitive Inhibition An inhibitor competes with a substrate for the binding site of an enzyme. As an enzyme-inhibitor complex does not contribute to product formation, it decreases the rate of product formation. Many competitive inhibitors have steric structures similar to substrates, and are referred to as substrate analogs. 

The search for selective means of inactivation, prompted by clinical considerations, has not been successful, although many substrate analogs act as powerful competitive inhibitors 8). On the other hand, irreversible inhibition may result from interaction with substrate analogs which cause labilization of the enzyme (see Section IV,B). Specific stimulation and inhibition by homologous antibodies is reviewed in Section V. 

Cidofovir is a cytosine nucleotide analog with in vitro activity against CMV, HSV-1, HSV-2, VZV, EBV, HHV-6, HHV-8, adenovirus, poxviruses, polyomaviruses, and human papillomavirus. In contrast to ganciclovir, phosphorylation of cidofovir to the active diphosphate is independent of viral enzymes. After phosphorylation, cidofovir acts both as a potent inhibitor of and as an alternative substrate for viral DNA polymerase, competitively inhibiting DNA synthesis and becoming incorporated into the viral DNA chain. Isolates with resistance to cidofovir have been selected in vitro these isolates tend to be cross-resistant with ganciclovir but retain susceptibility to foscamet. Clinically significant resistance to cidofovir has not been reported to date. 

Catalyst inhibition is traditionally associated with biocatalytic processes, but can also apply to homogeneous and heterogeneous catalysis. Competitive inhibition is analogous to competitive adsorption in gas/solid heterogeneous catalysis, where two molecules from the gas phase compete for the same active site on the catalyst surface. A competitive inhibitor is any chemical species I which can bind to the same site as the substrate, or to another site on the enzyme (an allosteric site). The overall reaction scheme is then given by Eqs. (2.58)-(2.60), where El indicates an enzyme-inhibitor complex. 

Enzyme activity may be inhibited by substances that inactivate the enzyme or occupy the active site of the enzyme before the substrate has a chance. As a result, the rate of transformation of the substrate to product is slowed. In competitive inhibition, similar substrates (or analogs) can bind to the same active site on the enzyme. Therefore, they compete with each other for the same active sites. This inhibition process is reversible and can be prevented or slowed by increasing the substrate concentration or by diluting the inhibitor in the solution. In this case, the enzyme already bound to the substrate is not inhibited. The effect of the competitive inhibitor (I) on the rate of enzyme reaction in Equation (5.129), Equation (5.130), Equation (5.131), and Equation (5.132) yields ... 

Examples of Rapid Reversible Inhibitors. Competitive inhibitors are often similar in structure to one of the substrates of the reaction they are inhibiting. Inhibitors of this type are sometimes called substrate analogs and their binding affinity (K ) usually approximates that of the substrate. One of the first reactions inhibited by a substrate analog was that catalyzed by succinate dehydrogenase (Equation 17.24). 

The three most common t3q>es of reversible inhibition occurring in enzymatic reactions are competitive, uncompetitive, and noncompetitive. (See Problem P7-12b) The enzyme molecule is analogous to the heterogeneous catalytic surface in that it contains active sites. When competitive Inhibition occurs, the substrate and inhibitor are usually similar molecules that compete for the same site on the enzyme. Uncompetitive inhibition occurs when the inhibitor deactivates the enzyme-substrate complex, usually by attaching itself to both tlie substrate and enzyme molecules of the complex. Noncompetitive inhibition occurs with enzymes containing at least two different types of sites. The inhibitor attaches to only one type of site and the substrate only to the other. Derivation of the rate laws for these three types of inhibition is shown on the CD-ROM. 

Cidofovir (Figure 24.4) is an antiviral cytidine nucleotide analog with inhibitory activity against HCMV and other herpes viruses. Cidofovir is first converted to an active diphosphate form by cellular enzymes. Antiviral effects of cidofovir are due to inhibition of viral DNA polymerase by the diphosphate metabolite (Neyts and De Clercq, 1994 Plosker and Noble, 1999 Scholar and Pratt, 2000). The diphosphate probably interacts with DNA polymerase either as an alternate substrate (incorporation at the 3 end or within the interior of the DNA chain) or as a competitive inhibitor (with respect to the normal substrate dCTP). Cidofovir inhibits HCMV DNA synthesis at intracellular concentrations 1000-fold lower than are required to inhibit cellular DNA synthesis (Neyts and De Clercq, 1994). For HSV-1 and HSV-2 corresponding concentrations are at least 50-fold lower. 

Methotrexate inhibits DNA synthesis by decreasing avail-ability of pyrimidine nucleotides. Methotrexate competitively inhibits the enzyme dihydrofolate reductase, thus decreasing the concentrations of the tetrahydrofolate essential to the methylation of the pyrimidine nucleotides and consequently the rate of pyrimidine nucleotide synthesis. Leucovorin, a folate analog, is used to rescue host cells from methotrexate inhibition as a synthetic substrate for dihydrofolate reductase, leucovorin administration allows resumption of tetrahydrofolate-dependent synthesis of pyrimidines and reinitiation of DNA synthesis. Methotrexate is a nonspecific cytotoxin, and prolongation of blood levels appropriate to killing tumor cells may lead to severe, unwanted cytotoxic effects such as myelosuppression, gastrointestinal mucositis, and hepatic cirrhosis. 

Several studies have shown that hyaluronidase is inhibited by Fe3+, Fe2+, Cu2+, Ag+, Hg2+, Zn2+, Cd2+, and Pb2+ salts [26,51,58,77,78]. Substrate analogs like chondroitin sulfate B, desulfated chondroitin sulfate B, dermatan sulfate, keratan sulfate, heparitin sulfate, and heparin were shown to be competitive inhibitors of hyaluronidase [14,54]. 

The three most common types of reversible inhibition occurring in enzymatic reaction.s are cotnpeiitive, uncompetitive, and noncompetitive. The enzyme molecule is analogous to a heterogeneous catalytic surface in that it contains active sites. When competitive inhibition occurs, the substrate and... 

In an ordered sequential mechanism where C is an alternate substrate for the second substrate, B, one observes competitive inhibition by C versus B and noncompetitive inhibition versus A. for the competitive inhibition is given by an equation analogous to Eq. (25) containing ia, and A in place of ib, b(c). and B. For the noncompetitive inhibition, is given by an equation analogous to Eq. (26), with KaKc K (e), and B replacing K, Ka,... 

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