a) Energy of activation
b) Net free energy change
c) Equilibrium concentration of substrate
d) Equilibrium concentration of product
Answer- The right answer is – a) Energy of activation. The enzymes decrease the energy of activation for a reaction and speed up the reaction by many folds. The thermodynamics of a reaction, such as the free energy change and the equilibrium concentrations of the substrate and product remain unchanged.
Case details- It is a case of Cardiac failure.
The renin-angiotensin-aldosterone system (RAAS) plays an important role in regulating blood volume and systemic vascular resistance, which together influence cardiac output and arterial pressure.
As the name implies, there are three important components to this system: 1) renin, 2) angiotensin, and 3) aldosterone. Renin, which is primarily released by the kidneys, stimulates the formation of angiotensin in blood and tissues, which in turn stimulates the release of aldosterone from the adrenal cortex. It is called a system because each part influences the other parts and all are necessary for the whole to function correctly.
Renin is a proteolytic enzyme that is released into the circulation primarily by the kidneys. Its release is stimulated by:
1) sympathetic nerve activation (acting via β1-adrenoceptors)
2) renal artery hypotension (caused by systemic hypotension or renal artery stenosis)
3) decreased sodium delivery to the distal tubules of the kidney.
When renin is released into the blood, it acts upon a circulating substrate, angiotensinogen, that undergoes proteolytic cleavage to form the decapeptide angiotensin I. Vascular endothelium, particularly in the lungs, has an enzyme, angiotensin converting enzyme (ACE), that cleaves off two amino acids to form the octapeptide, angiotensin II (AII), although many other tissues in the body (heart, brain, vascular) also can form AII (Figure-1).
Angiotensin I is able to alter the blood pressure to some degree, but it isn’t strong enough to cause large changes. Instead, most angiotensin I is converted to angiotensin II, a much more powerful hormone that does cause large changes in blood pressure. (This conversion is shut down by drugs called ACE Inhibitors, an important type of high blood pressure medication.)
Angiotensin II is a strong hormone, and can act directly on blood vessels to cause blood pressure increases. It also has another even more important function – stimulating the release of aldosterone. Aldosterone is a very powerful vasoconstrictor that causes large increases in blood pressure, but is more important because it can actually change the baseline filtering activity of the kidneys. Aldosterone causes the kidneys to retain both salt and water, which – over time – increases the amount of water in the body. This increase, in turn, raises blood pressure.
Figure-1- Renin angiotensin system.
Activation of the renin-angiotensin-aldosterone (RAA) system rapidly kicks in with heart failure, due to decreased renal perfusion caused by both a reduction in cardiac output and redistribution of blood away from nonessential organs (kidney). The kidney retains sodium and water in response to the perception of ineffective blood volume. The perception of decreased blood volume and the increase in sympathetic nervous activity stimulates renin release from the juxtaglomerular cells in the kidneys.
The compensatory mechanisms in heart failure eventually initiate a vicious cycle which leads to continued worsening and downward spiraling of the heart failure state. The peripheral vasoconstriction mediated by increased sympathetic activity, angiotensin II, and other possible mechanisms causes an increase in systemic vascular resistance or afterload. Afterload resists myocardial fiber shortening and further decreases cardiac output, which leads to further increases in sodium and water retention and sympathetic nervous activity (Figure-2).
Therapeutic manipulation of this pathway is very important in treating hypertension and heart failure. ACE inhibitors, Angiotensin II receptor blockers and aldosterone receptor blockers, for example, are used to decrease arterial pressure, ventricular afterload, blood volume and hence ventricular preload, as well as inhibit and reverse cardiac and vascular hypertrophy.
Mechanism of action of ACE inhibitors- Frequently prescribed ACE inhibitors include perindopril, captopril, enalapril, lisinopril, and ramipril. ACE inhibitors block the conversion of angiotensin I to angiotensin II. They act by competitive inhibition.
General Mechanism of Action of Enzymes
Enzymes are catalysts and increase the speed of a chemical reaction without themselves undergoing any permanent chemical change. They are neither used up in the reaction nor do they appear as reaction products.
The basic enzymatic reaction can be represented as follows
where E represents the enzyme catalyzing the reaction, S the substrate, the substance being changed, and P the product of the reaction.
Enzymes employ multiple mechanisms to facilitate catalysis. The mechanism of action of enzymes can be explained by two perspectives-
1) Thermodynamic changes
2) Processes at the active site
1) Thermodynamic changes - All enzymes accelerate reaction rates by providing transition states with a lowered G for formation of the transition states. However, they may differ in the way this is achieved.
A chemical reaction of substrate S to form product P goes through a transition state S‡ that has a higher free energy than does either S or P. (The double dagger denotes a thermodynamic property of the transition state).
The difference in free energy between the transition state and the substrate is called the Gibbs free energy of activation or simply the activation energy, symbolized by ∆G‡.
The activation energy barrier suggests how enzymes enhance reaction rate without altering ∆G of the reaction: enzymes function to lower the activation energy, or, in other words, enzymes facilitate the formation of the transition state. The combination of substrate and enzyme creates a new reaction pathway whose transition-state energy is lower than that of the reaction in the absence of enzyme (see Figure 3). The lower activation energy means that more molecules have the required energy to reach the transition state. Decreasing the activation barrier is analogous to lowering the height of a high-jump bar; more athletes will be able to clear the bar. The essence of catalysis is specific binding of the transition state.
Figure-3 Enzymes Decrease the Activation Energy. Enzymes accelerate reactions by decreasing ∆G, the free energy of activation.
2) Processes at the active site-Enzymes use various combinations of four general mechanisms to achieve dramatic catalytic enhancement of the rates of chemical reactions. These are as follows-
a) Catalysis by Bond Strain: In this form of catalysis, the induced structural rearrangements that take place with the binding of substrate and enzyme ultimately produce strained substrate bonds, which more easily attain the transition state. Enzymes that catalyze lytic reactions that involve breaking a covalent bond typically bind their substrates in a conformation slightly unfavorable for the bond that will undergo cleavage. The resulting strain stretches or distorts the targeted bond, weakening it and making it more vulnerable to cleavage.
b) Catalysis by Proximity and Orientation: For molecules to react, they must come within bond-forming distance of one another. The higher their concentration, the more frequently they will encounter one another and the greater will be the rate of their reaction. When an enzyme binds substrate molecules at its active site, it creates a region of high local substrate concentration. Enzyme-substrate interactions orient reactive groups and bring them into proximity with one another.
c) Catalysis Involving Proton Donors (Acids) and Acceptors (Bases): Other mechanisms also contribute significantly to the completion of catalytic events initiated by a strain mechanism, for example, the use of glutamate as a general acid catalyst (proton donor). The ionizable functional groups of aminoacyl side chains and (where present) of prosthetic groups contribute to catalysis by acting as acids or bases. Acid-base catalysis can be either specific or general. By “specific” we mean only protons (H3O+) or OH– ions. In specific acid or specific base catalysis, the rate of reaction is sensitive to changes in the concentration of protons but independent of the concentrations of other acids (proton donors) or bases (proton acceptors) present in solution or at the active site. Reactions whose rates are responsive to all the acids or bases present are said to be subject to general acid or general base catalysis.
d) Covalent Catalysis: In catalysis that takes place by covalent mechanisms, the substrate is oriented to active sites on the enzymes in such a way that a covalent intermediate forms between the enzyme or coenzyme and the substrate. One of the best-known examples of this mechanism is that involving proteolysis by serine proteases, which include both digestive enzymes (trypsin, chymotrypsin, and elastase) and several enzymes of the blood clotting cascade. These proteases contain an active site serine whose R group hydroxyl forms a covalent bond with a carbonyl carbon of a peptide bond, thereby causing hydrolysis of the peptide bond. Covalent catalysis introduces a new reaction pathway whose activation energy is lower—and therefore is faster—than the reaction pathway in homogeneous solution. The chemical modification of the enzyme is, however, transient. On completion of the reaction, the enzyme returns to its original unmodified state. Its role thus remains catalytic. Covalent catalysis is particularly common among enzymes that catalyze group transfer reactions.
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Which of the following statements is not true of competitive inhibitors ?
a) Vmax remains the same
b) Apparent Km in increased
c) Inhibitor is a structural analogue of the substrate
d) Inhibitor binds covalently to the enzyme
e) Increasing concentration of substrate can reverse the changes
Myasthenia gravis (MG) is a relatively rare autoimmune disorder of peripheral nerves in which antibodies form against acetylcholine (ACh) nicotinic postsynaptic receptors at the neuromuscular junction (NMJ). The basic pathology is a reduction in the number of ACh receptors (AChR) at the postsynaptic muscle membrane brought about by an acquired autoimmune reaction producing anti-AChR antibodies. MG is classified into 2 major clinical forms: ocular MG and generalized MG.
The reduction in the number of AChRs results in a characteristic pattern of progressively reduced muscle strength with repeated use and recovery of muscle strength after a period of rest. The bulbar muscles are affected most commonly and most severely, but most patients also develop some degree of fluctuating generalized weakness.
MG is one of the most treatable neurologic disorders. Pharmacologic therapy includes anticholinesterase medication and immunosuppressive agents. Anticholinesterase agents include pyridostigmine, neostigmine, and edrophonium while immunosuppressive agents include corticosteroids, Azathioprine, Cyclosporine A etc.
In the given case physostigmine is anticholinesterase medication that is known to act by competitive inhibition of Acetylcholinestrase. Acetyl choline is left undegraded.
In competitive inhibition, an enzyme can bind substrate (forming an ES complex) or inhibitor (EI) but not both (ESI). The competitive inhibitor resembles the substrate and binds to the active site of the enzyme (Figure-1). The substrate is thereby prevented from binding to the same active site.
A competitive inhibitor diminishes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate. The hallmark of competitive inhibition is that it can be overcome by a sufficiently high concentration of substrate. Under these conditions, the substrate “outcompetes” the inhibitor for the active site. However, the apparent value of Km is altered; the effect of a competitive inhibitor is to increase the apparent value of Km. As the value of [I] increases, the value of Km increases (see Figure-2). In the presence of a competitive inhibitor, an enzyme will have the same V max as in the absence of an inhibitor. The inhibitor does not bind covalently to the enzyme to bring about irreversible changes. Thus in the given problem, all statements are true of competitive inhibitors except (d).
Figure-1-The competitive inhibitor resembles the substrate and binds to the active site of the enzyme. The substrate is thereby prevented from binding to the same active site.
Figure-2- As the concentration of a competitive inhibitor increases, higher concentrations of substrate are required to attain a particular reaction velocity. A sufficiently high concentrations of substrate can completely relieve competitive inhibition.
A brief summary of enzyme inhibitors is as follows-
|Type of Inhibition||Effect on Maximum Reaction Velocity (Vmax) and Km||Reversible/ Irreversible||Examples|
(Inhibitor- a structural Analogue).
There is a competition between substrate and the inhibitor for the active site
|1) Vmax -Unchanged
2) Km Increased
|Reversible||1) HMG Co A Reductase- inhibited by Statins- used as cholesterol lowering drugs.
2) Epoxide Reductase – inhibited by Dicoumarol- used as an anticoagulant.
3) Dihydrofolate Reductase-inhibited by Methotrexate- used as anticancer drug.
4) Pteroyl Synthase- inhibited by PABA (Para- amino –benzoic –acid) used as antibiotic.
5) Angiotensin converting enzyme Inhibitor- inhibited by Captopril –used as an antihypertensive drug.
6) Succinate dehydrogenase- inhibited by Malonate acts as a poison.
7) Lactate dehydrogenase- inhibited by Oxamate- acts as a poison.
|Non competitive (Inhibitor binds at a site other than the active site)||1) Vmax- Decreased
2) Km -Unchanged
|Can be reversible or irreversible||1) Enolase is inhibited by Fluoride used for sample collection for glucose estimation.
2) PDH complex, Alpha ketoglutarate dehydrogenase complex, glyceraldehyde-3-P dehydrogenase(-SH group containing enzymes) are inhibited by Arsenate, Acts as a slow poison
3) Cytochrome oxidase- Inhibited by cyanide acts as a poison.
Also called Mechanism based inhibition
Inhibitor gets activated by host enzyme to inhibit the subsequent enzyme
|1) Vmax- Decreased
2) Km- Increased
|Irreversible||1)Inhibition of Xanthine oxidase by Allopurinol, used for the treatment of gout.
2) Inhibition of Aconitase by fluoroacetate- used as a rat poison
3) Inhibition of Thymidylate synthase by 5-FU (5-Fluoro Uracil) used as an anticancer drug
4) Inhibition of Ornithine decarboxylase by Di fluoro methyl ornithine(DFMO)
5) Mono Amine Oxidase is inhibited by Deprenyl used to treat Parkinson disease and depression.
|Allosteric Inhibition (Binding of the inhibitor alters either the affinity of the enzyme for its substrate or the reaction velocity is decreased||1) V max is decreased in V type enzymes
2) Km is increased in K type enzymes
|Reversible||Most of them are physiological inhibitors.
1)ATP and Citrate are allosteric inhibitors of PFK-1
2) Fr 2,6 bisphosphate is an allosteric inhibitor of Fr1,6 bisphosphatase enzyme.
|Feed back inhibition, also called Product inhibition The product of the reaction pathway inhibits the key regulatory enzyme.||1) Vmax-Decreased
2) Km- constant
|Reversible depending upon the need of the product||1) Inhibition of HMG Co A Reductase by Mevalonate(Immediate product) and cholesterol(Final product)
2) Inhibition of Aspartate transcarbamoylase UTP and CTP, the products of this pathway.
|Un competitive inhibition Inhibitor binds to the enzyme substrate complex||Vmax and Km both are decreased||Ir- reversible||Inhibition of placental alkaline phosphatase by Phenyl Alanine|
After the clotted blood was spun down in the test tube, the serum was visibly hemolyzed owing to difficulty in obtaining the specimen. The physical examination of the child was unremarkable.
Why is the normal range of ALP higher in children than in adults?
What is the significance of a hemolyzed serum sample and the elevated serum ALP?
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