Clinical Enzymology

Richard A. McPherson MD, MSc , in Henry's Clinical Diagnosis and Management by Laboratory Methods , 2022

Competitive Inhibition

Competitive inhibition occurs when the inhibitor binds at the same site as the substrate. The molecular basis for the binding of competitive inhibitors at the active site is that the substrate and the inhibitor are structurally similar, with the result that the enzyme is "deceived" into recognizing and binding the inhibitor. Examples of competitive inhibition include the inhibition of trypsin by α-1-antitrypsin, chymotrypsin by α-1-antichymotrypsin, dihydrofolate reductase by the chemotherapeutic agent methotrexate, and the Krebs cycle enzyme succinic dehydrogenase by malonate. In serum assays, urea inhibits LD, and inorganic phosphate inhibits alkaline phosphatase. Fluoride ion competitively inhibits the glycolytic enzyme enolase. In some reactions, the product of the reaction, which may be structurally similar to the substrate, may competitively inhibit the reaction, a phenomenon known as end-product inhibition. A variation of this phenomenon can occur occasionally in which the buildup of the end-product can reverse the forward reaction, regenerating the substrate. Not infrequently, drugs are used as competitive inhibitors of targeted enzymes. For example, as discussed later, a major target of antihypertensive drugs is angiotensin-converting enzyme (ACE).

Enzymes and Nucleic Acids

Hyone-Myong Eun , in Enzymology Primer for Recombinant DNA Technology, 1996

i. Competitive inhibition.

Competitive inhibition is usually caused by substances that are structurally related to the substrate, and thus combine at the same binding site as the substrate. The bindings are exclusive to each other, forming either an enzyme–substrate (ES) or an enzyme–inhibitor (EI) complex but not a ternary complex (EIS) ( Scheme 1.3, Fig. 1.3). This type of inhibition can be completely overcome by high substrate concentrations and thus does not affect the V. The K m is increased by a factor of (1 + [I]/K i).

SCHEME 1.3.

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Role of the Liver in Metabolism

John W. Baynes PhD , in Medical Biochemistry , 2019

Induction and competitive inhibition of cytochrome P-450 enzymes underpin mechanisms of drug interactions

Hepatic synthesis of cytochromes P-450 is induced by certain drugs and other xenobiotic agents that increase the rate of the phase I reactions. Conversely, drugs that form a relatively stable complex with a particular cytochrome P-450 inhibit the metabolism of other drugs that are normally substrates for that cytochrome. For instance, CYP1A2 metabolizes, among others, caffeine and theophylline. It can be inhibited by grapefruit juice, which contains a substance known as naringin, or by the antibiotic ciprofloxacin. When a person takes any of the inhibitory substances, normal substrates for CYP1A2 are metabolized more slowly, and their plasma levels increase.

The dose of the immunosuppressant ciclosporin may need to be reduced by up to 75% if the patient also takes the antifungal drug ketoconazole (seeWilkinson inFurther Reading) to avoid adverse clinical reactions.

The drugs that induce induction or repression of CYP3A enzymes often act through the nuclear receptor mechanism. They combine with nuclear receptors (i.e., in the case of CYP3A4, the pregnane X receptor [PXR]), which then form heterodimers with retinoid X receptors (Chapter 14). Such complexes upregulate CYP3 synthesis by binding to response elements in the gene promoter.

Enzymes and Enzyme Regulation

N.V. Bhagavan , Chung-Eun Ha , in Essentials of Medical Biochemistry (Second Edition), 2015

Reversible Inhibition by Reaction Products

Competitive inhibition can occur in freely reversible reactions owing to accumulation of products. Even in reactions that are not readily reversible, a product can function as an inhibitor when an irreversible step precedes the dissociation of the products from the enzyme. In the alkaline phosphatase reaction, in which hydrolysis of a wide variety of organic monophosphate esters into the corresponding alcohols (or phenols) and inorganic phosphates occurs, the inorganic phosphate acts as a competitive inhibitor. Both the inhibitor and the substrate have similar enzyme-binding affinities (i.e., K m and Ki are of the same order of magnitude).

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Toxic alcohol poisoning

POLLY E. PARSONS MD , in Critical Care Secrets , 2019

3. How does competitive inhibition play a role in the management of toxic alcohol ingestions?

All of the toxic alcohols use ADH as the initial enzyme in metabolism. However, it is clinically useful in coingestions with the toxic alcohols because the administration of fomepizole may be safely delayed until ethanol is metabolized to a concentration of approximately 100 mg/dL. Slowing of ADH is accomplished clinically by competitive inhibition, either by ethanol or fomepizole, a medication that serves as a direct competitive inhibitor of ADH.

Enzymes I: General Properties, Kinetics, and Inhibition

N.V. BHAGAVAN , in Medical Biochemistry (Fourth Edition), 2002

Inhibition of Folate Synthesis

Competitive inhibition of a biosynthetic step in folate synthesis accounts for the antimicrobial action of sulfonamides, which are structural analogues of p-aminobenzoic acid (PABA):

PABA is used by bacteria in the synthesis of folic acid (pteroylglutamic acid), which functions as a coenzyme in one-carbon transfer reactions that are important in amino acid metabolism, in the synthesis of RNA and DNA, and thus in cell growth and division. Sulfonamides inhibit the bacterial enzyme responsible for incorporation of PABA into 7,8-dihydropteroic acid (Figure 6-7) and lead to the inhibition of growth (bacteriostasis) of a wide range of gram-positive and gram-negative microorganisms. Microorganisms susceptible to sulfonamides are those which synthesize their own folic acid or which cannot absorb folic acid derived from the host. Sulfonamides, however, have no effect on host cells (or other mammalian cells) that require preformed folic acid.

FIGURE 6-7. Folate biosynthetic pathways in bacteria. The incorporation of p-aminobenzoic acid into 7,8-dihydropteroic acid is competitively inhibited by sulfanilamide.

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Antiadrenergic Agents☆

Enrico Agabiti-Rosei , in Encyclopedia of Endocrine Diseases (Second Edition), 2019

α-Adrenoceptor Antagonists (α-Blockers)

Competitive inhibition of α-adrenoceptors by appropriate antagonists predominantly causes vasodilatation and a reduction of blood pressure, particularly in hypertensive patients. Doxazosin and prazosin are the prototypes of α 1-blockers. They are selective for the postsynaptic α1-adrenoceptor subtype: This means that presynaptic α2-adrenoceptors are not blocked, thus preventing the enhanced release of NE from the sympathetic nerve endings (Fig. 2). Nonselective, older α1-blockers, such as phentolamine or phenoxybenzamine, are no longer used because they are poorly tolerated.

Doxazosin and prazosin are moderately effective antihypertensive drugs. Orthostatic hypotension, and sometimes headache, and reflex tachycardia are well-known adverse reactions, which are caused by vasodilation (also in the venous blood vessels). A few newer α-adrenoceptor antagonists display a certain degree of selectivity for the α1A-receptor in the smooth muscle of the prostate. These agents cause relaxation of this smooth muscle tissue and hence facilitate urinary flow in patients with benign prostate hyperplasia (BPH). Alfuzosin, terazosin, and tamsulosin are examples of such agents. Silodosin is an α-blocker more uroselective and therefore may induce less postural hypotension, but ejaculation disfunction may be even more frequent.

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Advances in Radiation Biology

Walter Sauerbier , in Advances in Radiation Biology, 1976

B Specific Binding of RNA Polymerase to Various DNA Templates Is Not Affected Even at Very High UV Doses

Competitive inhibition of synthesis to RNA from nonirradiated DNA by the presence of UV-irradiated DNA was utilized as a measure of the binding of RNA polymerase to UV-irradiated T4 DNA ( Sauerbier et al., 1970). These studies employed the following concept: The rate of RNA synthesis with a mixture of DNA templates, one-half of which is nonirradiated and the other half irradiated, should be one-half the sum of the individual rates of RNA synthesis with either template, (R O + R UV)/2, provided that UV irradiation has not altered the binding of RNA polymerase to DNA and that the reaction is saturated with each template. Reduced polymerase binding to UV-irradiated DNA would be indicated by resultant rates higher than (R O + R UV)/2. (Here, R O stands for the rate of RNA synthesis with the nonirradiated DNA template and R UV for the rate observed with the irradiated template.) As Fig. 2 shows, binding of E. coli RNA polymerase to DNA of bacteriophage T4 is not measurably affected by UV doses up to approximately 1000 ergs/mm2. This dose is equivalent to about 150 lethal hits to T4vx (Harm, 1963), or about 220 thymine dimers in the early region of the T4 genome (Sauerbier, 1964; Sauerbier et al., 1970), or about three to four phage-lethal hits per T4 scripton comprised of an average of three to four genes (Stahl et al., 1970; Sauerbier et al., 1970; Hercules and Sauerbier, 1973; O'Farrell and Gold, 1973). Clearly, loss of binding of RNA polymerase to UV-irradiated DNA contributes little, if at all, to the loss of viability of T4.

Fig. 2. In vitro rates of RNA synthesis with nonirradiated T4 DNA, UV-irradiated T4 DNA and mixtures of nonirradiated and UV-irradiated T4 DNA. Curve A: (•) kinetics of [3H] ATP incorporation with 34 μg/ml unirradiated T4 DNA; (○) with 34 μg/ml DNA present at the onset of incubation and additional 34 μg/ml added 8 min later. Curve B: (▪) same as curve A (•) but with 940 ergs/mm2 irradiated T4 DNA; (□) same as curve A (○) but with 940 ergs/mm2 irradiated T4 DNA. Curve E: one-half the sum of the rates of synthesis with unirradiated (A) and with 940 ergs/mm2 irradiated T4 DNA (B). Curve C: (Δ) RNA synthesis with 34 μg/ml unirradiated DNA present at the onset of incubation and 34 μg/ml of 940 ergs/mm2 unirradiated DNA added 8 min later. Curve D: (▴) same as curve C, except that 34 μg/ml of 940 ergs/mm2 irradiated DNA were added first and the unirradiated 34 Mg/ml were added 8 min later. The specific activity of [3H] ATP was 1 Ci/mole, and the concentration of RNA polymerase 10.15 μg/ml. Ordinate gives the nmoles ATP incorporated in 0.2-ml aliquots. Abscissa gives the time of incubation at 37°C.

 From Sauerbier et al. (1970) with permission of Elsevier Publishing Company. Copyright © 1970

Inspection of Fig. 2 shows a resultant rate of RNA synthesis that is actually less than one-half the sum of the rates with either template. This has been interpreted as a slowdown in release of RNA polymerase from the UV-irradiated template DNA and not as an increased binding to UV-irradiated DNA (Sauerbier et al., 1970). Since this interpretation agrees with other observations on the transcription of UV-irradiated DNA [no loss of RNA polymerase binding (Ishihama and Kameyama, 1967; Chamberlin and Ring, 1970), no effect on the rate of RNA chain initiation during the first 10 min of polymerization (Sauerbier et al., 1970), and effective recycling of RNA polymerase (Michalke and Bremer, 1969; Sauerbier et al., 1970)], it seems to be correct.

No loss of binding of E. coli RNA polymerase to E. coli DNA has been reported by Ishihama and Kameyama (1967) and no loss of T7 RNA polymerase binding to T7 DNA up to 80,000 ergs/mm2 was reported by Chamberlin and Ring (1973). These latter authors argued that the number of polymerase binding sites does not increase as a consequence of UV irradiation to T7 DNA. In contrast to the observations made with UV-irradiated bacterial and bacteriophage DNA, formation of new, unproductive binding sites for E. coli RNA polymerase by UV irradiation of calf thymus DNA has been repeatedly reported (Hagen et al., 1964; Zimmermann et al., 1965). Since the initiation of transcription on calf thymus DNA occurs nonspecifically (Burgess et al., 1969) at single-strand breaks (Hagen et al., 1970), the UV effects on binding of polymerase and on RNA chain initiation with this particular template should not be generalized.

Systematic investigations of RNA polymerase binding to UV-irradiated DNA templates, involving several types of RNA polymerase and several types of DNA templates and covering a wide dose range, are still lacking, although the DNA binding assay to nitrocellulose filters via RNA polymerase (Jones and Berg, 1966; Hagen et al., 1970) should make direct binding observations experimentally quite feasible (at least for RNA polymerases which bind strongly to the DNA template).

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Pharmacodynamics

Elaine M Aldred BSc (Hons), DC, Lic Ac, Dip Herb Med, Dip CHM , ... Kenneth Vall , in Pharmacology, 2009

Enzyme Inhibition

Most enzyme receptor sites are not completely specific (there is some structural leeway given the number of combinations possible and the mobility of the protein) and a relatively similarly shaped molecule might be able to achieve a 'close fit'. This creates competition for molecules of a similar shape and the original molecule might find itself unable to find a binding site because it is already occupied. Many drugs are designed to take advantage of this phenomenon.

The various ways in which enzyme function can be affected are not dissimilar to the ways receptor function can be affected. These principles are worth bearing in mind when looking at chemicals that act directly on receptor sites.

• Competitive Inhibition

•

Competitive inhibition [ Figure 19.2(i)] is reversible: another molecule competes with the normal substrate and takes its place in the site.

•

However, when the normal substrate concentration exceeds that of the competing molecule, the situation is more favourable and the normal substrate replaces the competing molecule.

•

While the competing molecule is in place it blocks the normal action of the enzyme.

•

Competitive inhibition can be reversed by increasing the substrate concentration.

• Non-competitive Inhibition

•

Non-competitive inhibition [ Figure 19.2(ii)] is reversible.

•

The inhibitor, which is not a substrate, attaches itself to another part of the enzyme, thereby changing the overall shape of the site for the normal substrate so that it does not fit as well as before, which slows or prevents the reaction taking place.

•

This type of inhibition decreases the turnover rate of an enzyme rather than interfering with the amount of substrate binding to the enzyme. The reaction is slowed rather than stopped. Non-competitive inhibition, therefore, cannot be increased by increasing the substrate.

• Irreversible Inhibition

•

The inhibitor becomes covalently linked or bound to the enzyme so tightly that is very difficult to detach it from the enzyme [Figure 19.2(iii); see Chapter 3 'Bonds found in biological chemistry', p. 13].

•

The situation cannot be reversed.

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Clinical Pharmacokinetics and Drug Interactions

Nilanjan Saha , in Pharmaceutical Medicine and Translational Clinical Research, 2018

6.5.2.3 DDI during drug metabolism

The competitive inhibition of CYP enzymes occurs when inhibitor and substrate compete for the same binding site on the enzyme, resulting in a DDI. In this type of interaction, the inhibition is reversible. It depends on the relative concentrations of substrate and the inhibitor. Some of the inhibitors of CYP3A4 that act by this mechanism of inhibition include azole antifungal agent ketoconazole. In the noncompetitive mechanism, the inhibitor and substrate do not compete for the same site on the CYP enzyme. When a ligand binds to the allosteric site, the conformational changes of the active site occur, leading to reduced binding of the substrate. Many drugs are noncompetitive inhibitors of CYP enzymes, like omeprazole and lansoprazole, and cimetidine. The inhibition is reversed when new enzymes are synthesized after the inhibitor drug is withdrawn.

Drug interactions involving enzyme induction are less common than inhibition-based drug interactions but are clinically important. Environmental pollutants as well as lipophilic drugs can result in induction of CYP enzymes. The increased synthesis of CYP enzyme proteins induced by one drug speed up the metabolism and clearance of the other drug in DDI. The most commons enzyme inducers are rifampicin, phenytoin, and carbamazepine.

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