Inhibition, of allosteric enzymes

Binding of a reversible inhibitor to an enzyme is rapidly reversible and thus bound and unbound enzymes are in equilibrium. Binding of the inhibitor can be to the active site, or to a cofactor, or to some other site on the protein leading to allosteric inhibition of enzyme activity. The degree of inhibition caused by a reversible inhibitor is not time-dependent the final level of inhibition is reached almost instantaneously, on addition of inhibitor to an enzyme or enzyme-substrate mixture. 

The development of the concept of allosteric inhibition of enzymes began in the early 1950s but, surprisingly, not with studies on enzymes. It was discovered that addition of an amino acid to a culture of bacteria Escherichia colt)... 

The inhibition of certain enzymes by specific metabolites is an important element in the regulation of intermediary metabolism and most often occurs with cooperative enzymes that are regulated allosterically. Inhibition of enzymes that obey the Michaelis-Menten equation, noncooperative enzymes, is more commonly used by pharmacists to alter a patient s metabolism. Reversible inhibition of noncooperative enzymes is classified into three groups which can be distinguished kinetically and which have different mechanisms and effects when administered. The classes are called competitive, uncompetitive, and noncompetitive inhibition. Mixed inhibition also occurs. In all these types of inhibition, the inhibitor (usually a small molecule) binds reversibly and rapidly with the enzyme. 

Substances that do not target the active site but display inhibition by allosteric mechanisms are associated with a lower risk of unwanted interference with related cellular enzymes. Allosteric inhibition of the viral polymerase is employed in the case of HIV-1 nonnucleosidic RT inhibitors (NNRTl, see chapter by Zimmermann et al., this volume) bind outside the RT active site and act by blocking a conformational change of the enzyme essential for catalysis. A potential disadvantage of targeting regions distant from the active site is that these may be subject to a lower selective pressure for sequence conservation than the active site itself, which can lower the threshold for escape of the virus by mutation. 

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the NAD(P)H-dependent reduction of 5,10-methylenetetrahydrofolate (CH2-THF) to 5-methyltetrahydrofolate (CH3-THF). CH3-THF then serves as a methyl donor for the synthesis of methionine. The MTHFR proteins and genes from mammalian liver and E. coli have been characterized,12"15 and MTHFR genes have been identified in S. cerevisiae16 and other organisms. The MTHFR of E. coli (MetF) is a homotetramer of 33-kDa subunits that prefers NADH as reductant,12 whereas mammalian MTHFRs are homodimers of 77-kDa subunits that prefer NADPH and are allosterically inhibited by AdoMet.13,14 Mammalian MTHFRs have a two-domain structure the amino-terminal domain shows 30% sequence identity to E. coli MetF, and is catalytic the carboxyterminal domain has been implicated in AdoMet-mediated inhibition of enzyme activity.13,14... 

A good example of allosteric inhibition is given by hexokinase (HK) isoenzymes of muscle. The product of the HK reaction, glucose-6-P allosterically inhibits the enzyme, so matching the phosphorylation of glucose to its overall metabolism, helps to regulate... 

Severe combined immunodeficiency arises from inhibition of lymphocyte proliferation because B and T cells are particularly sensitive to allosteric inhibition of which of the following enzymes of purine nucleotide metabolism ... 

The answer is D. Impaired immune function in severe combined immunodeficiency (SCID) is the direct result of blocked DNA synthesis due to inadequate supplies of de-oxyribonucleotides in B and T cells. This effect arises by dATP-induced allosteric inhibition of ribonucleotide reductase, which catalyzes reduction of the 2 -hydroxyl groups on ADP and GDP to form dADP and dCDP. The ultimate cause of many cases of SCID is adenosine deaminase deficiency, which leads to accumulation of dATP and consequent inhibition of ribonucleotide reductase. Although the other enzymes mentioned are also involved in purine nucleotide metabolism, their deficiencies do not lead to SCID. 

Antimetabolite inhibitors of DNA synthesis act by the competitive or allosteric inhibition of a number of different enzymes participating in purine or pyrimidine biosynthesis. Actually, some such compounds interfere with as many as 10-12 different enzymes— although admittedly to a different degree. 

The PDH complex of mammals is strongly inhibited by ATP and by acetyl-CoA and NADH, the products of the reaction catalyzed by the complex (Fig. 16-18). The allosteric inhibition of pyruvate oxidation is greatly enhanced when long-chain fatty acids are available. AMP, CoA, and NAD+, all of which accumulate when too little acetate flows into the citric acid cycle, allosterically activate the PDH complex. Thus, this enzyme activity is turned off when ample fuel is available in the form... 

The flow of intermediates through metabolic pathways is controlled by 1bir mechanisms 1) the availability of substrates 2) allosteric activation and inhibition of enzymes 3) covalent modification of enzymes and 4) induction-repression of enzyme synthesis. This scheme may at first seem unnecessarily redundant however, each mechanism operates on a different timescale (Figure 24.1), and allows the body to adapt to a wde variety of physiologic situations. In the fed state, these regulatory mechanisms ensure that available nutrients are captured as glycogen, triacylglycerol, and protein. 

Figure 11-3 Feedback inhibition of enzymes involved in the biosynthesis of threonine, isoleucine, methionine, and lysine in E. coli. These amino acids all arise from L-aspartate, which is formed from oxaloacetate generated by the biosynthetic reactions of the citric acid cycle (Fig. 10-6). Allosteric inhibition. Q Repression of transcription of the enzyme or of its synthesis on ribosomes.

Pyruvate kinase catalyzes the third irreversible step in glycolysis. It is activated by fructose 1,6-bisphosphate. ATP and the amino acid alanine allosterically inhibit the enzyme so that glycolysis slows when supplies of ATP and biosynthetic precursors (indicated by the levels of Ala) are already sufficiently high. In addition, in a control similar to that for PFK (see above), when the blood glucose concentration is low, glucagon is released and stimulates phosphorylation of the enzyme via a cAMP cascade (see Topic J7). This covalent modification inhibits the enzyme so that glycolysis slows down in times of low blood glucose levels. 

We start our analysis of the TCA cycle kinetics by examining the predicted steady state production of NADH as a function of the NAD and ADP concentrations. From Equation (6.31) we see that there can be no net flux through the TCA cycle when concentration of either NAD or ADP, which serve as substrates for reactions in the cycle, is zero. Thus when the ratios [ATP]/[ADP] and [NADH]/[NAD] are high, we expect the TCA cycle reaction fluxes to be inhibited by simple mass action. In addition, the allosteric inhibition of several enzymes (for example inhibition of pyruvate dehydrogenase by NADH and ACCOA) has important effects. 

Phosphofructokinase, Phosphofructokinase is the most important control site in the mammalian glycolytic pathway (Figure 16.1 S). High levels of ATP allosterically inhibit the enzyme (a 340-kd tetramer). ATP binds to a specific regulatory site that is distinct from the catalytic site. The binding of ATP lowers the enzyme s affinity for fructose 6-phosphate. Thus, a high concentration of ATP converts the hyperbolic binding curve of fructose... 

Research into the control of glycolytic rate in muscle has revealed that enzyme activity may also be controlled by reversible formation of enzyme-F-actin complexes. F-actin is a polymer of actin molecules and makes up one of the two muscle filaments that participate in muscle contraction. It can be shown that enzymes, such as PK, readily bind to F-actin filaments under conditions of low ionic strength in vitro (Chan et al., 1986). Extrapolation of the conditions in the test tube to conditions found in cells suggests that a significant proportion of PK may be bound in vivo (Brooks and Storey, 1991 a). In the case of PK, binding decreases the enzyme activity by increasing the Km value for PEP. F-actin, therefore, acts like ATP and alanine in allosterically inhibiting the enzyme. 

Ai with other antimetabolites, acquired resistance is a major obstacle. The most common mechanism of 6-MP resistance is deficiency or complete lack of the activating enzyme, HGPRT. Another mechanism of resistance is increased particulate alkaline phosphatase activity. Other potential mechanisms for resistance to 6-MP include (1) decreased drug transport (2) alteration in allosteric inhibition of ribosylamine 5-phosphate synthase (3) altered recognition of DNA breaks and mismatches induced by 6-MP and (4) increased activity of multidrug resistance protein 5, which exports nucleoside analogs. 

Fig. 22.14. Regulation of PFK-1 by AMP, ATP and fructose-2,6-bisP. A. AMP and fructose 2,6-bisphosphate activate PFK-1. B. ATP increases the rate of the reaction at low concentrations, but allosterically inhibits the enzyme at high concentrations.

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