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.Please help "Biochemistry for Medics" by CLICKING ON THE ADVERTISEMENTS above!
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
Figure- Difficulty in opening the eyelids in myashenia gravis
The answer is D-Inhibitor binds covalently to the enzyme.
Myasthenia gravis (MG)
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 Acetylcholinesterase. Acetylcholine 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 Vmax 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|
1) Inhibitor- a structural analogue
2) 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 Dicumarol-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.
|Noncompetitive 1) Inhibitor binds at a site other than the active site.
2) Not a structural analogue
|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, which acts as a poison.
|Suicidal inhibition, also called Mechanism based inhibition
Inhibitor gets activated by host enzyme to inhibit the subsequent enzyme
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-Fluorouracil)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) Vmax 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.
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 by
UTP and CTP, the products of this pathway.
|Un- competitive inhibition Inhibitor binds to the enzyme substrate complex||Vmax and Km -both are decreased||Irreversible||Inhibition of placental alkaline phosphatase- by Phenylalanine|
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Characteristics of active site
1) The active site takes the form of a cleft or pocket which is formed by groups that come from different parts of the amino acid sequences. The residues far apart in the sequence may interact more strongly than adjacent residues in the amino acid sequence. Amino acids near to one another in the primary structure are often sterically constrained from adopting the structural relations necessary to form the active site.
2) The active sites of multimeric enzymes are located at the interface between subunits and recruit residues from more than one monomer.
3) It takes up a relatively small part of the total volume of an enzyme. Most of the amino acid residues in an enzyme are not in contact with the substrate. Nearly all enzymes are made up of more than 100 amino acid residues, the “extra” amino acids serve as a scaffold to create the three-dimensional active site from amino acids that are far apart in the primary structure. In many proteins, the remaining amino acids also constitute regulatory sites, sites of interaction with other proteins, or channels to bring the substrates to the active sites.
4) Substrates are bound to enzymes by multiple weak attractions. The noncovalent interactions in Enzyme Substrate (ES) complex are much weaker than covalent bonds.The electrostatic interactions, hydrogen bonds, van der Waals forces, and hydrophobic interactions mediate reversible interactions of biomolecules.
5) The specificity of binding depends on the precisely defined arrangement of atoms in an active site. Because the enzyme and the substrate interact by means of short-range forces that require close contact, a substrate must have a matching shape to fit into the site.
Chemistry of active site for Enzyme substrate binding
Two models have been proposed to explain how an enzyme binds its substrate: the lock-and –key model and the induced-fit model.
1) Lock-and-Key Model of Enzyme-Substrate Binding– In this model, the active site of the unbound enzyme is complementary in shape to the substrate.
Figure-1 showing the lock and key model of Enzyme substrate model
“lock and key model” accounted for the exquisite specificity of enzyme-substrate interactions, the implied rigidity of the enzyme’s active site failed to account for the dynamic changes that accompany catalysis.
2) Induced-Fit Model of Enzyme-Substrate Binding In this model, the enzyme changes shape on substrate binding. The active site forms a shape complementary to the substrate only after the substrate has been bound.When a substrate approaches and binds to an enzyme they induce a conformational change, a change analogous to placing a hand (substrate) into a glove (enzyme).
Figure-2- Showing induced fit model of enzyme substrate binding
The induced fit model has been amply confirmed by biophysical studies of enzyme motion during substrate binding. Besides explaining the specificity, it also explains the regulation of enzyme activity and the dynamic changes occurring at the active site.Please help "Biochemistry for Medics" by CLICKING ON THE ADVERTISEMENTS above!
The catalytic activity of many enzymes depends on the presence of small molecules termed cofactors, although the precise role varies with the cofactor and the enzyme. Such an enzyme without its cofactor is referred to as an apoenzyme; the complete, catalytically active enzyme is called a holoenzyme.
Cofactors can be subdivided into two groups: metals and small organic molecules
- Most common cofactor are metal ions . (Some sources also limit the use of the term “cofactor” to inorganic substances).
- Cofactors that are small organic molecules are called coenzymes.
- If tightly bound, the cofactors are called prosthetic groups
- Loosely bound cofactors serve functions similar to those of prosthetic groups but bind in a transient, dissociable manner either to the enzyme or to a substrate.
- They are more like co substrates because they bind to and are released from the enzyme just as substrates and products are.
Tightly integrated into the enzyme structure by covalent or non-covalent forces. It can be organic or inorganic (metal ions) e.g.
◦ Pyridoxal phosphate
◦ Flavin mononucleotide( FMN)
◦ Flavin adenine dinucleotide(FAD)
◦ Thiamin pyrophosphate (TPP)
Metals are the most common prosthetic groups
◦ Metal ions – Co, Cu, Mg, Mn, Zn, Fe
Role of metal ions
- Enzymes that contain tightly bound metal ions are termed – Metalloenzymes.
- Enzymes that require metal ions as loosely bound cofactors are termed as metal-activated enzymes
Metal ions facilitate
◦ Binding and orientation of the substrate
◦ Formation of covalent bonds with reaction intermediates
◦ Interact with substrate to render them more electrophilic or nucleophilic
Examples of Metallo enzymes- (Table-1)
Fe+++ or Fe++
Cu++ or Cu+
Propionyl CoA carboxylase
Metal activated /Ion activated enzymes
In a few enzyme-controlled reactions, it is the presence of certain ions that can increase the reaction rate. Ions may combine with the enzyme or the substrate. The ion binding makes the formation of an enzyme-substrate complex happen more easily, because it can affect the charge distribution or the end shape of the complex.
Amylase catalyses the breakdown of maltose molecules. This enzyme will function properly only if chloride ions are present. Without the chloride ions, amylase cannot catalyse the reaction
Co-enzymes serve as recyclable shuttles—or group transfer agents—that transport many substrates from their point of generation to their point of utilization.
- The water-soluble B vitamins supply important components of numerous coenzymes
- Chemical moieties transported by coenzymes include hydrogen atoms or hydride ions, methyl groups (folates), acyl groups (coenzyme A), and oligosaccharides (dolichol).
Examples of Coenzymes- (Table-2)
|Nicotine adenine dinucleotide||NAD+ – Derived from niacin||Electron (hydrogen atom)||Lactate dehydrogenase|
|Nicotine adenine dinucleotide phosphate||NADP+ – niacin derivative||Electron (hydrogen atom)||Glutamate dehydrogenase|
|Flavin adenine dinucleotide||FAD – riboflavin (vit. B2) derivative||electron (hydrogen atom)||Monoamine oxidase|
|Coenzyme A||CoA||Acyl groups||Acetyl CoA carboxylase|
|Thiamine pyrophosphate||Thiamine (vit. B1)||Aldehydes||Pyruvate dehydrogenase Complex|
|Pyridoxal phosphate||Pyridoxine (vit B6)||amino and many other||Transaminases, Decarboxylases, Glycogen phosphorylase|
|5′-Deoxyadenosyl cobalamine||vit. B12||alkyl groups||Methylmalonyl mutase|
|Tetrahydrofolate||Folic acid||One carbon compounds||Thymidylate synthase|
The water-soluble B vitamins supply important components of numerous coenzymes. Several coenzymes contain, in addition, the adenine, ribose, and phosphoryl moieties of AMP or ADP (Table–2). Nicotinamide is a component of the redox coenzymes NAD and NADP, whereas riboflavin is a component of the redox coenzymes FMN and FAD. Pantothenic acid is a component of the acyl group carrier coenzyme A. As its pyrophosphate, thiamin participates in decarboxylation of α-keto acids and folic acid and cobamide coenzymes function in one-carbon metabolism.Please help "Biochemistry for Medics" by CLICKING ON THE ADVERTISEMENTS above!
A complete, balanced set of enzyme activities is of fundamental importance for maintaining homeostasis.
Numerous factors affect the reaction rate
The kinetic theory—also called the collision theory—of chemical kinetics states that for two molecules to react they must –
(1) approach within bond-forming distance of one another, or “collide”; and
(2) must possess sufficient kinetic energy to overcome the energy barrier for reaching the transition state.
It therefore follows that anything that increases the frequency or energy of collision between substrates will increase the rate of the reaction in which they participate.
Raising the temperature increases the kinetic energy of molecules. Increasing the kinetic energy of molecules also increases their motion and therefore the frequency with which they collide. This combination of more frequent and more highly energetic and productive collisions increases the reaction rate. A ten degree Centigrade rise in temperature will increase the activity of most enzymes by 50 to 100%. The Q10, or temperature coefficient, is the factor by which the rate of a biologic process increases for a 10 °C increase in temperature. For the temperatures over which enzymes are stable, the rates of most biologic processes typically double for a 10 °C rise in temperature (Q10 = 2).
Figure-1- showing the effect of temperature on enzyme catalyzed reaction.
The reaction rate increases with temperature to a maximum level, then abruptly declines with further increase of temperature (Figure-1). Because most animal enzymes rapidly become denatured at temperatures above 40oC, most enzyme determinations are carried out somewhat below that temperature. Some enzymes lose their activity when frozen.The optimal temperatures of the enzymes in higher organisms rarely exceed 50 °C, while enzymes from thermophilic bacteria found in hot springs, for instance, may still be active at 100 °C.
Changes in the rates of enzyme-catalyzed reactions that accompany a rise or fall in body temperature constitute a prominent survival feature for “cold-blooded” life forms such as lizards or fish, whose body temperatures are dictated by the external environment. However, for mammals and other homoeothermic organisms, changes in enzyme reaction rates with temperature assume physiologic importance only in circumstances such as fever or hypothermia.
2) Hydrogen Ion Concentration
The rate of almost all enzyme-catalyzed reactions exhibits a significant dependence on hydrogen ion concentration. Most intracellular enzymes exhibit optimal activity at pH values between 5 and 9 (Figure-2).The pH optimum—i. e., the pH value at which enzyme activity is at its maximum—is often close to the pH value of the cells (i. e., pH 7). However, there are also exceptions to this. For example, the proteinase pepsin , which is active in the acidic gastric lumen, has a pH optimum of 2, while other enzymes (at least in the test tube) are at their most active at pH values higher than 9 (Figure-3)
When the activity is plotted against pH, a bell-shaped curve is usually obtained (Figure-2)
Figure-2- Showing the effect of pH on enzyme catalyzed reaction
Figure-3- Except for Pepsin, acid phosphatase and alkaline phosphatase, most enzyme have optimum pH between 5 to 9
The relationship of activity to hydrogen ion concentration reflects the balance between enzyme denaturation at high or low pH and effects on the charged state of the enzyme, the substrates, or both. For enzymes whose mechanism involves acid-base catalysis, the residues involved must be in the appropriate state of protonation for the reaction to proceed. The binding and recognition of substrate molecules with dissociable groups also typically involves the formation of salt bridges with the enzyme. The most common charged groups are the negative carboxylate groups and the positively charged groups of protonated amines. Gain or loss of critical charged groups thus will adversely affect substrate binding and thus will retard or abolish catalysis.
3) Substrate concentration
The frequency with which molecules collide is directly proportionate to their concentrations. For two different molecules A and B, the frequency with which they collide will double if the concentration of either A or B is doubled. If the concentrations of both A and B are doubled, the probability of collision will increase fourfold.
For a typical enzyme, as substrate concentration is increased; vi increases until it reaches a maximum value Vmax (Figure -4)When further increases in substrate concentration do not further increase Vmax the enzyme is said to be “saturated” with substrate. If a curve is plotted,the shape of the curve that relates activity to substrate concentration (Figure-4) is hyperbolic.
At any given instant, only substrate molecules that are combined with the enzyme as an ES complex can be transformed into product. Second, the equilibrium constant for the formation of the enzyme-substrate complex is not infinitely large. Therefore, even when the substrate is present in excess, (points A and B of Figure), only a fraction of the enzyme may be present as an ES complex. At points A or B, increasing or decreasing [S] therefore will increase or decrease the number of ES complexes with a corresponding change in vi. The rate of reaction is substrate dependent (First order reaction)- Figure-4
At point C (Figure), essentially all the enzyme is present as the ES complex. Since no free enzyme remains available for forming ES, further increases in [S] cannot increase the rate of the reaction . Reaction rate therefore becomes independent of substrate concentration (Zero order reaction).
Figure-4- Showing the relationship of reaction rate with substrate concentration
4) Effect of Enzyme concentration
In order to study the effect of increasing the enzyme concentration upon the reaction rate, the substrate must be present in an excess amount; i.e., the reaction must be independent of the substrate concentration. Any change in the amount of product formed over a specified period of time will be dependent upon the level of enzyme present. Graphically this can be represented as:
Figure-5- Showing the effect of increasing or decreasing enzyme concentration on reaction rate
These reactions are said to be “zero order” because the rates are independent of substrate concentration, and are equal to some constant k. The formation of product proceeds at a rate which is linear with time. The addition of more substrate does not serve to increase the rate. In zero order kinetics, allowing the assay to run for double time results in double the amount of product (Figure-5)
The amount of enzyme present in a reaction is measured by the activity it catalyzes. The relationship between activity and concentration is affected by many factors such as temperature, pH, etc. An enzyme assay must be designed so that the observed activity is proportional to the amount of enzyme present in order that the enzyme concentration is the only limiting factor. It is satisfied only when the reaction is zero order.
Enzyme activity is generally greatest when substrate concentration is unlimiting.
5) Effect of product concentration
The enzyme activity declines when the products start accumulating. This is called product or feed back inhibition. Under certain conditions reverse reaction may be favored forming back the substrate.
6) Effect of activators and Coenzymes
The activity of certain enzymes is greatly dependent on metal ion activators and coenzymes. Vitamins act as coenzymes in a variety of reactions.
7) Effect of modulators and Inhibitors
The enzyme activity is reduced in the presence of an inhibitor and on the other hand the enzyme activity may be increased in the presence of a positive modifier.
In typical enzyme-catalyzed reactions, reactant and product concentrations are usually hundreds or thousands of times greater than the enzyme concentration. Consequently, each enzyme molecule catalyzes the conversion to product of many reactant molecules. In biochemical reactions, reactants are commonly known as substrates. The catalytic event that converts substrate to product involves the formation of a transition state, and it occurs most easily at a specific binding site on the enzyme. This site, called the catalytic site of the enzyme, has been evolutionarily structured to provide specific, high-affinity binding of substrate(s) and to provide an environment that favors the catalytic events. The complex that forms, when substrate(s) and enzyme combine, is called the enzyme substrate (ES) complex. Reaction products arise when the ES complex breaks down releasing free enzyme.
Between the binding of substrate to enzyme, and the reappearance of free enzyme and product, a series of complex events must take place. At a minimum an ES complex must be formed; this complex must pass to the transition state (ES*); and the transition state complex must advance to an enzyme product complex (EP). The latter is finally competent to dissociate to product and free enzyme. The series of events can be shown thus:
E + S <——> ES <——> ES* <——> EP <——> E + P
The kinetics of simple reactions like that above were first characterized by biochemists Michaelis and Menten. The concepts underlying their analysis of enzyme kinetics continue to provide the cornerstone for understanding metabolism today, and for the development and clinical use of drugs aimed at selectively altering rate constants and interfering with the progress of disease states.
The Michaelis-Menten equation is a quantitative description of the relationship among the rate of an enzyme- catalyzed reaction [v1], the concentration of substrate [S] and two constants, Vmax and km (which are set by the particular equation). The symbols used in the Michaelis-Menten equation refer to the reaction rate [v1], maximum reaction rate (V max), substrate concentration [S] and the Michaelis-Menten constant (km).
The Michaelis-Menten equation can be used to demonstrate that at the substrate concentration that produces exactly half of the maximum reaction rate, i.e. ½ V max the substrate concentration is numerically equal to Km. This fact provides a simple yet powerful bioanalytical tool that has been used to characterize both normal and altered enzymes, such as those that produce the symptoms of genetic diseases.
Thus, the Michaelis constant, km is the substrate concentration at which V1 is half the maximal velocity (Vmax /2) attainable at a particular concentration of enzyme. km thus has the dimensions of substrate concentration.
The dependence of initial reaction velocity on [S] and Km may be illustrated by evaluating the Michaelis-Menten equation under three conditions.
(1) When [S] is much less than km, the term km + [S] is essentially equal tokm. Replacing Km + [S] with Km reduces equation to
Since V max and km are both constants, their ratio is a constant (k). In other words, when [S] is considerably below km, V max is proportionate to k[S]. The initial reaction velocity therefore is directly proportionate to [S].
(2) When [S] is much greater than km, the term km + [S] is essentially equal to [S]. Replacing km + [S] with [S] reduces equation to
Thus, when [S] greatly exceeds km, the reaction velocity is maximal (V max) and unaffected by further increases in substrate concentration.
(3) When [S] = km
Equation states that when [S] equals km, the initial velocity is half-maximal. Equation also reveals that km is—and may be determined experimentally from—the substrate concentration at which the initial velocity is half-maximal.
Figure-6-Plot of substrate concentration versus reaction velocity
The key features of the plot are marked by points A, B and C. At high substrate concentrations the rate represented by point C the rate of the reaction is almost equal to V max and the difference in rate at nearby concentrations of substrate is almost negligible. If the Michaelis-Menten plot is extrapolated to infinitely high substrate concentrations, the extrapolated rate is equal to V max When the reaction rate becomes independent of substrate concentration, or nearly so, the rate is said to be zero order. (Note that the reaction is zero order only with respect to this substrate. If the reaction has two substrates, it may or may not be zero order with respect to the second substrate). The very small differences in reaction velocity at substrate concentrations around point C (near V max) reflect the fact that at these concentrations almost all of the enzyme molecules are bound to substrate and the rate is virtually independent of substrate, hence zero order. At lower substrate concentrations, such as at points A and B, the lower reaction velocities indicate that at any moment only a portion of the enzyme molecules are bound to the substrate. In fact, at the substrate concentration denoted by point B, exactly half the enzyme molecules are in an ES complex at any instant and the rate is exactly one half of V max At substrate concentrations near point A the rate appears to be directly proportional to substrate concentration, and the reaction rate is said to be first order.
A Linear Form of the Michaelis-Menten Equation Is Used to determine km & V max
The direct measurement of the numeric value of V max and therefore the calculation of km often requires impractically high concentrations of substrate to achieve saturating conditions. A linear form of the Michaelis-Menten equation circumvents this difficulty and permits V max and km to be extrapolated from initial velocity data obtained at less than saturating concentrations of substrate. Starting with equation,
Equation is the equation for a straight line, y = ax + b, where y = 1/vi and x = 1/[S]. A plot of 1/vi as y as a function of 1/[S] as x therefore gives a straight line whose y intercept is 1/ V max and whose slope is km / V max. Such a plot is called a double reciprocal or Lineweaver-Burk plot (Figure-7). Setting the y term of equation equal to zero and solving for x reveals that the x intercept is -1/Km.
Figure- 7-showing A Lineweaver-Burk Plot Plots of 1/v versus 1/[S] yield straight lines having a slope of Km/Vmax and an intercept on the ordinate at 1/Vmax.
An alternative linear transformation of the Michaelis-Menten equation is the Eadie-Hofstee transformation: and when v/[S] is plotted on the y-axis versus v on the x-axis, the result is a linear plot with a slope of –1/Km and the value Vmax/Km the intercept on the y-axis and as the Vmax intercept on the x-axis.
Both the Lineweaver-Burk and Eadie-Hofstee transformation of the Michaelis-Menten equation are useful in the analysis of enzyme inhibition.
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Classification of enzymes- More than 2000 different enzymes are currently known. The commonly used names for most enzymes describe the type of reaction catalyzed, followed by the suffix -ase. For example, dehydrogenases remove hydrogen atoms, proteases hydrolyze proteins, and isomerases catalyze rearrangements in configuration. Modifiers may precede the name to indicate the substrate (xanthine oxidase), the source of the enzyme (pancreatic ribonuclease), its regulation (hormone-sensitive lipase) etc.
To address ambiguities, the International Union of Biochemists (IUB) developed an unambiguous system of enzyme nomenclature in which each enzyme has a unique name and code number that identify the type of reaction catalyzed and the substrates involved. Enzymes are grouped into six classes:
1) The oxidoreductases (class 1) catalyze the transfer of reducing equivalents(Hydrogen and electrons)from one redox system to another.
2) The transferases (class 2) catalyze the transfer of other groups from one molecule to another. Oxidoreductases and transferases generally require coenzymes
3) The hydrolases (class 3) hydrolases cause cleavage of bond using water
4) Lyases (class 4, often also referred to as“synthases”) catalyze reactions involving either the cleavage or formation of chemical bonds, with double bonds either arising or disappearing.(See figure- reversible reaction is shown). Cleavage of bond does not require water.
5) The isomerases (class 5) move groups within a molecule, without changing the gross composition of the substrate.
6) The ligation reactions catalyzed by ligases (“synthetases,” class 6) are energy-dependent and are therefore always coupled to the hydrolysis of nucleoside triphosphates(See figure)
Each enzyme is entered in the Enzyme Catalogue with a four-digit Enzyme Commission number (EC number). The first digit indicates membership of one of the six major classes. The next two indicate subclasses and subsubclasses. The last digit indicates where the enzyme belongs in the subsubclass.
For example, The IUB name of hexokinase is ATP:D-hexose 6-phosphotransferase E.C. 220.127.116.11. This name identifies hexokinase as a member of class 2 (transferases), subclass 7 (transfer of a phosphoryl group), sub-subclass 1 (alcohol is the phosphoryl acceptor), and “hexose-6” indicates that the alcohol phosphorylated is on carbon six of a hexose. However, it is still called as hexokinase.
Figure- Showing the classification of enzymes with examples of each class of enzymes
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|Type of Inhibition||Effect on Maximum Reaction Velocity(Vmax)||Effect on Km(Affinity of Enzyme for its substrate)||Reversible/ Irreversible||Examples|
|Competitive Inhibition (Inhibitor- a structural Analogue).
There is a competition between substrate and the inhibitor for the active site
|Unchanged||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 convertase 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)||Decreased||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.
4)Cyclooxygenase is inhibited by Aspirin-used as an anti inflammatory, analgesic and antipyretic drug (Inhibits formation of prostaglandins)
|Suicidal inhibition, Also called Mechanism based inhibition
Inhibitor gets activated by host enzyme to inhibit the subsequent enzyme
|Decreased||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 Transpeptidase by Penicillin- used as an antibiotic
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||V max is decreased in V type enzymes||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.||Decreased||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
by UTP and CTP, the products of this pathway.
|Un competitive inhibition Inhibitor binds to the enzyme substrate complex||Decreased||Decreased||Ir- reversible||Inhibition of placental alkaline phosphatase by Phenyl Alanine|
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Isoenzymes are enzymes that catalyze identical chemical reactions but are composed of different amino acid sequences. They are sometimes referred to as isozymes. Isoenzymes are produced by different genes and are not redundant despite their similar functions. They occur in many tissues throughout the body and are important for different developmental and metabolic processes.
Isoenzymes are useful biochemical markers and can be measured in the bloodstream to diagnose medical conditions. Isoenzymes can be differentiated from one another using gel electrophoresis. In gel electrophoresis, isoenzyme fragments are drawn through a thick gel by an electric charge. Each isoenzyme has a distinct charge of its own because of its unique amino acid sequence. This enables gel electrophoresis to separate the fragments into bands for identification. Some clinically important isoenzymes are as follows:
1) Creatine Kinase(CK, CPK) is an enzyme found primarily in the heart and skeletal muscles, and to a lesser extent in the brain but not found at all in liver and kidney. Small amounts are also found in lungs, thyroid and adrenal glands. Significant injury to any of these structures will lead to a measurable increase in CK levels. It is not found in red blood cells and its level is not affected by hemolysis.
Normal Value– serum activity varies from 10-50 IU/L at 30°C.
Elevations are found in:
- Myocardial infarction
- Crushing muscular trauma
- Any cardiac or muscle disease, but not myasthenia gravis or multiple sclerosis
- Brain injury
After myocardial infarction– serum value is found to increase within hours, reaches a peak level in 24- 30 hours and returns to normal level in 2-4 days (usually in 72 hours). CK is a sensitive indicator in the early stages of myocardial ischemia. No increase in activity is found in heart failure and coronary insufficiency. In acute MI, CPK usually rises faster than SGOT and returns to normal faster than the SGOT.
There are three Isoenzymes. Measuring them is of value in the presence of elevated levels of CK or CPK to determine the source of the elevation. Each iso enzyme is a dimer composed of two protomers‘M’ (for muscles) and ‘B’( for Brain). These isoenzymes can be separated by, Electrophoresis or by Ion exchange Chromatography. The three possible iso enzymes are;
|Isoenzyme||Electrophoretic mobility||Tissue of origin||Mean percentage in blood|
- Normal levels of CK/CPK are almost entirely MM, from skeletal muscle.
- Elevated levels of CK/CPK resulting from acute myocardial infarction are about half MM and half MB. Myocardial muscle is the only tissue that contains more than five percent of the total CK activity as the CK2 (MB) isoenzyme.
- Following an attack of acute myocardial infarction, this isoenzyme appears within 4 hours following onset of chest pain, reaches a peak of activity at approximately 24 hours and falls rapidly. MB accounts for 4.5- 20 % of the total CK activity in the plasma of the patients with recent myocardial infarction and the total isoenzyme is elevated up to 20-folds above the normal.
- Atypical Isoenzymes– In addition to the above three isoenzymes two atypical iso enzymes of CPK have been reported. They are; Macro CK(CK-Macro) and Mitochondrial CK(CK-Mi).
o Macro CK(CK-Macro)- It is formed by the aggregation of CK-BB with immunoglobulins usually with IgG but sometimes Ig A.It may also be formed by complexing CK-MM with lipoproteins. No specific disease has been found to be associated with this isoenzyme.
o Mitochondrial CK(CK-Mi)- It is presentbound to the exterior surface of the inner mitochondrial membrane of muscle,liver and brain. It can exist in dimeric form or as oligomeric aggregates having molecular weight of approximately 35,000. It is only present in serum when there is extensive tissue damage causing breakdown of mitochondrial membrane and cell membrane. Thus its presence in serum indicates severe illness and cellular damage. It is not related with any specific disease states but it has been detected in certain cases of malignant tumors.
2) Aspartate amino Transferase (AST)
It is also called as Serum Glutamate Oxalo acetate Transaminase (SGOT). The level is significantly elevated in Acute MI.
Normal Value- 0-41 IU/L at 37°C
In acute MI- Serum activity rises sharply within the first 12 hours, with a peak level at 24 hours or over and returns to normal within 3-5 days. The rise depends on the extent of infarction. Re- infarction results in secondary rise of SGOT.
- Levels> 350 IU/L are due to massive Infarction(Fatal),
- > 150 IU/L are associated with high mortality and levels
- < 50IU/L are associated with low mortality.
Other diseases– The rise in activity is also observed in muscle and hepatic diseases. These can be well differentiated from simultaneous estimations of other enzyme activities like SGPT etc, which do not show and rise in activity in Acute MI.
3) Alanine amino transferase (ALT)- Also called serum Glutamate pyruvate transaminase.
Normal serum level ranges between 0-45 IU/L at37oC.
Very high values are seen in Acute hepatitis,toxic or viral in origin. Both ALT and AST rise but ALT> AST. Moderate increase may be seen in chronic liver diseases such as Cirrhosis and Malignancy in liver. A sudden fall in AST level in hepatitis signifies bad prognosis.
4) Lactate dehydrogenase (LDH)
Lactate dehydrogenase catalyzes the reversible conversion of pyruvate and lactate. LDH is essential for anaerobic respiration. When oxygen levels are low,LDH converts pyruvate to lactate, providing a source of muscular energy.
Normal level- 55-140IU/L at 30°C. The levels in the upper range are generally seen in children. LDH level is 100 times more inside the RBCs than in plasma, and therefore minor amount of hemolysis results in false positive result.
In Acute MI-The serum activity rises within 12 to 24 hours, attains a peak at 48 hours (2 to 4 days) reaching about 1000 IU/L and then returns gradually to normal from 8 th to 14 th day. The magnitude of rise is proportional to the extent of myocardial infarction. Serum LDH elevation may persist for more than a week after CPK and SGOT levels have returned to normal levels.
Other diseases-The increase in serum activity of LDH is also seen in hemolytic anemia, hepatocellular damage, muscular dystrophies,carcinoma, leukemias, and any condition which causes necrosis of the body cells. Since the total LDH is increased in many diseases, so the study of Isoenzymes of LDH is of more significance.
Iso enzymes of LDH
LDH enzyme is tetramer with 4 subunits. The subunit may be either H(Heart) or M(Muscle) polypeptide chains. These two chains are the product of 2 different genes. Although both of them have the same molecular weight, there are minor amino acid variations.There can be 5 possible combinations; H4, H3M1, H2M2, H1M3. M4, these are 5 different types of isoenzymes seen in all individuals.
|No. of Isoenzyme||Subunit make up of isoenzyme||Electrophoretic mobility at pH8.6||Activity at 60°for 30 minutes||Tissue origin||Percentage in human serum(Mean)|
|LDH-1||H4||Fastest||Not destroyed||Heart muscle||30%|
Normally LDH- 2(H3M1) level in blood is greater than LDH-1, but this pattern is reversed in myocardial infarction, this is called ‘flipped pattern’. These iso enzymes are separated by cellulose acetate electrophoresis at pH 8.6.
5) Alkaline phosphatase (ALP )-is an enzyme that removes phosphate groups from organic or inorganic compounds in the body. It is present in a number of tissues including liver, bone, intestine,and placenta. The activity of ALP found in serum is a composite of isoenzymes from those sites and, in some circumstances, placental or Regan isoenzymes. The optimum pH for enzyme action varies between 9-10. It is a zinc containing metalloenzyme and is localized in the cell membranes (ectoenzyme). It is associated with transport mechanism in the liver, kidney and intestinal mucosa.
Normal serum Level- of ALP ranges between 40-125 IU/L. In children the upper level of normal value may be more, because of increased osteoblastic activity.
Total Alkaline Phosphatase (ALP)
Serum ALP is of interest in the diagnosis of 2 main groups of conditions-hepatobiliary diseases and bone diseases associated with increased osteoblastic activity.
Mild increase is observed in pregnancy, due to production of placental enzyme.
A moderate rise in ALP activity occurs inhepatic diseases such as infective hepatitis, alcoholic hepatitis or hepatocellular carcinoma. Moderate elevation of ALP may also be seen in other disorders such as Hodgkin’s disease, congestive heart failure, ulcerative colitis, regional enteritis, and intra-abdominal bacterial infections.
ALP elevations tend to be more marked (more than 3-fold) in extra hepatic biliary obstructions (eg, by stone or cancer of the head of the pancreas) than in intrahepatic obstructions, and the more complete the obstruction, the greater the elevation. With obstruction, serumALP activities may reach 10 to 12 times the upper limit of normal, returning to normal upon surgical removal of the obstruction. The ALP response to cholestatic liver disease is similar to the response of gamma-glutamyltransferase (GGT), but more blunted. If both GGT and ALP are elevated, a liver source of the ALP is likely.
The response of the liver to any form of biliary tree obstruction is to synthesize more ALP. The main site of new enzyme synthesis is the hepatocytes adjacent to the biliary canaliculi.
ALP also is elevated in disorders of the skeletal system that involve osteoblast hyperactivity and bone remodeling, suchas Paget’s disease rickets and osteomalacia, fractures, and malignant tumors.
Among bone diseases, the highest level o f ALP activity is encountered in Paget’s disease, as a result of the action of the osteoblastic cells as they try to rebuild bone that is being resorbed by the uncontrolled activity of osteoclasts. Values from 10 to 25 times the upper limit of normal are not unusual.
Only moderate rises are observed in osteomalacia, while levels are generally normal in osteoporosis. In rickets, levels 2 to 4 times normal may be observed. Primary and secondary hyperparathyroidism are associated with slight to moderate elevations of ALP;the existence and degree of elevation reflects the presence and extent of skeletal involvement.
Very high enzyme levels are present in patients with osteogenic bone cancer.
A considerable rise in ALP is seen in children following accelerated bone growth.
Patients over age 60 can have a mildly elevated alkaline phosphatase (1–1½ times normal), while individuals with blood types O and B can have an elevation of the serum alkaline phosphatase after eating a fatty meal due to the influx of intestinal alkaline phosphatase into the blood.
Electrophoresis is considered the most useful single technique for ALP isoenzyme analysis. By starch gel electrophoresis at pH 8.6 , at least 6 isoenzyme bands can be visualized.
1) Hepatic ALP isoenzyme- moves fastest towards the anode and occupies the same position as Alpha 2 globulins. It is associated with biliary epithelium and is elevated in cholestatic processes. Various liver diseases (primary or secondary cancer, biliary obstruction) increase the liver isoenzyme.
2) Bone isoenzyme- It closely follows the hepatic enzyme and occupies the beta region. Osteoblastic bone tumors and hyperactivity of osteoblasts involved in bone remodeling (eg, Paget’s disease)increase the bone isoenzyme. Paget’s disease leads to a striking, solitary elevation of bone ALP.
3) Placental isoenzyme follows bone isoenzyme. It is heat stable isoenzyme and increases during last six weeks of pregnancy.
4) The intestinal isoenzyme-is the slowly moving band and follows the placental isoenzyme. It may be increased in patients with cirrhosis and in individuals who are blood group O or B secretors. Increased levels are also sen in patients undergoing hemodialysis.
Atypical ALP isoenzymes (Oncogenic markers)-In addition to 4 major isoenzymes, 2 more abnormal fractions are seen associated with tumors. These are Regan and Nagao isoenzymes. They are also called “Carcino placental ALP”, as they resemble placental isoenzymes.
Regan isoenzyme is elevated in various carcinomas of breast, lungs, colon and ovaries. Highest positivity is observed in carcinoma of ovary and uterus.
Rise in Nagao isoenzyme is observed in metastatic carcinoma of pleural surfaces and adenocarcinoma of pancreas and bile duct.Please help "Biochemistry for Medics" by CLICKING ON THE ADVERTISEMENTS above!