Allosteric inhibition

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) 

7 Discovery of reversible phosphorylation in the regulation of enzyme activity 

In the 1940s Carl and Gertrude Cori isolated and purified an active form (phosphorylase a) and an inactive form (phosphorylase b) of an enzyme from muscle. Phosphorylase b is activated by AMP (see page 64). In 1955, Fischer Krebs found an enzyme that catalysed the conversion of phosphorylase b to phosphorylase a, together with hydrolysis of ATP to ADP. Thus it appeared to bring about phosphorylation of the enzyme. The enzyme was termed phosphorylase b kinase, was partially purified and the interconversion was established as 

This suggested that the inactivating enzyme was a phosphatase and a protein phosphatase was purified, which catalysed the following reaction  

The existence of two forms of phosphorylase with different catalytic activities, which were capable of being enzymatically interconverted, suggested that the two forms might be involved in regulation of glycogenolysis in muscle tissue. However, in the early studies, whenever phosphorylase was assayed in extracts of muscle it was always found to be in the a form. This was the case even in extracts prepared from 

---

Inhibition of a regulatory enzyme by a feedback inhibitor does not conform to any normal inhibition pattern, and the feedback inhibitor F bears little structural similarity to A, the substrate for the regulatory enzyme. F apparently acts at a binding site distinct from the substrate-binding site. The term allosteric is apt, because F is sterically dissimilar and, moreover, acts at a site other than the site for S. Its effect is called allosteric Inhibition. 

Acetyl-CoA is a potent allosteric effector of glycolysis and gluconeogenesis. It allosterically inhibits pyruvate kinase (as noted in Chapter 19) and activates pyruvate carboxylase. Because it also allosterically inhibits pyruvate dehydrogenase (the enzymatic link between glycolysis and the TCA cycle), the cellular fate of pyruvate is strongly dependent on acetyl-CoA levels. A rise in... 

The metabolic control is exercised on certain key regulatory enzymes of a pathway called allosteric enzymes. These are enzymes whose catalytic activity is modulated through non-covalent binding of a specific metabolite at a site on the protein other than the catalytic site. Such enzymes may be allosterically inhibited by ATP or allosterically activated by ATP (some by ADP and/or AMP). 

A decreased glycolytic rate has been proposed as a cause of muscle fatigue and related to pH inhibition of glycolytic enzymes. Decreasing pH inhibits both phosphorylase kinase and phosphofructokinase (PFK) activities. PFK is rate determining for glycolytic flux and therefore must be precisely matched to the rate of ATP expenditure. The essential characteristic of PFK control is allosteric inhibition by ATP. This inhibition is increased by H and PCr (Storey and Hochachka, 1974 ... 

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. 

Koch U, Naqes F (2006) Allosteric inhibition of the hepatitis C virus NS5B RNA dependent RNA polymerase. Infect Disord Drug Targets 6 31 1... 

Fig. 3.15 Model for allosteric inhibition of a protein-DNA complex by a polyamide-inter-calator conjugate. (Top) The GCN4 homodimer (yellow) is displaced by the intercalating moiety (green) of the polyamide conjugate. Blue and red spheres represent pyrrole and imidazole amino acids, respectively. The blue diamond represents / -alanine. (Bottom, left) Hydrogen-bonding model of an eight-ring hairpin polyamide-intercalator conjugate...

Fechter, E. j. and P. B. Dervan. Allosteric inhibition of protein-DNA complexes by polyamide-intercalator conjugates. J. Am. Chem. Soc. 2003, 125, 8476-8485. 

To refer to the kinetics of allosteric inhibition as competitive or noncompetitive with substrate carries misleading mechanistic implications. We refer instead to two classes of regulated enzymes K-series and V-se-ries enzymes. For K-series allosteric enzymes, the substrate saturation kinetics are competitive in the sense that is raised without an effect on V. For V-series allosteric enzymes, the allosteric inhibitor lowers... 

Zhang X-Y, Bishop AC (2007) Site-specific incorporation of allosteric-inhibition sites in a protein tyrosine phosphatase. J Am Chem Soc 129 3812-3813... 

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... 

The mechanism of control of PFK-1 is mainly allosteric inhibition by ATP, citrate, long chain fatty acids and activation by AMP and F2,6-bisP. With the exception ofF2,6-bisP, the named regulators all indicate the fuel or energy status of the cell. Control by citrate helps to synchronise the rates of glycolysis and the TCA cycle. 

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. 

Reversible inhibition in which the inhibitor binds to a specific site on the enzyme that is remote to the active site, i.e. a distinct binding site, is known as allosteric inhibition. It is of considerable physiological importance and is considered in detail below. 

Reversible inhibitors are potentially less damaging. In the presence of a reversible inhibitor, the enzyme activity decreases, but to a constant level as equilibrium is reached. The enzyme activity reflects the lower level of enzyme available for catalysis. We can subdivide the reversible inhibition into three types, i.e. competitive, non-competitive, and allosteric inhibition. 

The third type of inhibition is called allosteric inhibition, and is particularly important in the control of intermediary metabolism This refers to the ability of enzymes to change their shape (tertiary and quaternary structure, see Section 13.3) when exposed to certain molecules. This sometimes leads to inhibition, whereas in other cases it may actually activate the enzyme. The process allows subtle control of enzyme activity according to an organism s demands. Further consideration of this complex phenomenon is outside our immediate needs. 

Most enzyme inhibitors act reversibly—i. e., they do not cause any permanent changes in the enzyme. However, there are also irreversible inhibitors that permanently modify the target enzyme. The mechanism of action of an inhibitor—its inhibition type—can be determined by comparing the kinetics (see p.92) of the inhibited and uninhibited reactions (B). This makes it possible to distinguish competitive inhibitors (left) from noncompetitive inhibitors (right), for example. Allosteric inhibition is particularly important for metabolic regulation (see below). 

PFK-1 is subject to allosteric inhibition by ATP, citrate, and phospho-enolpyruvate, all of which are elevated when the cell has a high level of energy reserves. 

The reaction is allosterically inhibited by high concentrations of AMP, an indicator of an energy-deficient state of the cell. 

The unphosphorylated form of PDH also is subject to direct allosteric inhibition by NADH and acetyl CoA. 

Isocitrate dehydrogenase is allosterically inhibited by NADH, an indicator of the availability of high levels of energy. 

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. 

---