Enzymes Theories of enzyme substrate interaction
2 Theories of enzyme substrate interaction
. There are two proposed methods by which enzymes bind to their substrate molecules:
1- Lock & key model
2-Induced fit model
3 Theories of enzyme substrate interaction 1-lock & key model (Template hypothesis for enzyme action): The substrate fits to binding site of enzyme as a key fits into proper lock . Enzyme & substrate possess specific complementary geometric & rigid shapes fit exactly into one another. This explains enzyme specificity, but fails to explain the stabilization of the transition state & allosteric regulation & inhibition. 4 1-lock & key model :
5 Theories of enzyme substrate interaction 2-Induce fit model More flexible model than lock & key model. Enzymes are flexible structures The active site modified as the [S] interacts with the anzyme (active site of enzyme become complementary to S) Model explains enzyme specificty & stabilization of transition state, regulation & inhibition.
6 2-Induce fit model
7 Enzyme Inhibition
8 Enzyme inhibitors:
Any substance that can diminish the velocity of an enzyme-catalyzed reaction.
9 Types of inhibition
Irreversible Reversible inhibitors inhibitors
10 Types of inhibition
1-Irreversible inhibitors :
Bind to enzymes through covalent bond (inhibited enzyme does not regain activity upon dilution of enzyme-inhibitor complex)
11 Type of inhibition
2-Reversible inhibitors:
Bind to enzymes through noncovalent bonds, thus dilution of the enzyme–inhibitor complex results in dissociation of the reversibly bound inhibitor, and recovery of enzyme activity.
12 Reversible inhibitors
2. Non competitive inhibition
3. Uncompeptitive inhibition
13 1-Competitive inhibition
The inhibitor and substrate compete for the same active site on the enzyme as a result of similarity in structure.
The enzyme substrate complex will be broken down to products (E+S ES E+P) where as enzyme inhibitor complex;(EI) will not be broken down to product. products.
14 1-Competitive inhibition
15 16 Kinetics of Competitive inhibition
Effect on Vmax:
. The effect of a competitive inhibitor is reversed by increasing [S]. . At a sufficiently high substrate concentration,
the reaction velocity reaches the Vmax observed in the absence of inhibitor (Vmax not changed)
17 Kinetics of Competitive inhibition
Effect on Km:
. A competitive inhibitor increases the
apparent Km for a given substrate. . This means that, in the presence of a competitive inhibitor, more substrate is
needed to achieve ½Vmax.
18 Kinetics of Competitive inhibition Effect on the Lineweaver-Burk plot: . Shows a characteristic Lineweaver-Burk plot in which the plots of the inhibited and uninhibited
reactions intersect on the y-axis at 1/Vmax (Vmax is unchanged). . The inhibited and uninhibited reactions show different x-axis intercepts, indicating that the
apparent Km is increased in the presence of the competitive.
19 Kinetics of Competitive inhibition
20 Examples of competitive inhibitors
Malonate that competes with succinate and inhibits the action of succinate dehydrogenase to produce fumarate in the Krebs cycle.
21 Examples of competitive inhibitors
Antihyperlipidemic agents competitively inhibits the first committed step in cholesterol synthesis. This reaction is catalyzed by HMG-CoA reductase. • Statin drugs, such as atorvastatin (Lipitor) and simvastatin (Zocor), are structural analogs of the natural substrate for this enzyme, and compete effectively to inhibit HMG-CoA reductase. • By doing so, they inhibit de novo cholesterol synthesis, thereby lowering plasma cholesterol levels.
22 Statins
23 Examples of competitive inhibitors
Allopurinol (used in treatment of gout) inhibits Xanthine oxidase by competing with Uric acid precursors for the active site on the enzyme. • This competition blocks the conversion of these precursors and of hypoxanthine and xanthine, to uric acid and result in lower serum urate levels.
24 25 Examples of competitive inhibitors
The antibiotic sulfanilamide is similar in structure to para-aminobenzoic acid (PABA), an intermediate in the biosynthetic pathway for folic acid. • Sulfanilamide can competitively inhibit the enzyme that has PABA as it's normal substrate by competitively occupying the active site of the enzyme.
26 27 2-Noncompetitive inhibition
• Noncompetitive inhibition occurs when the inhibitor and substrate bind at different sites on the enzyme.
• The noncompetitive inhibitor can bind either free enzyme or the ES complex, thereby preventing the reaction from occurring.
28 Noncompetitive inhibition
29 30 Kinetics of Noncompetitive inhibition
Effect on Vmax:
. Noncompetitive inhibition cannot be overcome by increasing the concentration of substrate. Thus, noncompetitive inhibitors decrease the
apparent Vmax of the reaction.
31 Kinetics of Noncompetitive inhibition
Effect on Km:
. Noncompetitive inhibitors do not interfere with the binding of substrate to enzyme.
Thus, the enzyme shows the same Km in the presence or absence of the noncompetitive inhibitor.
32 Kinetics of Noncompetitive inhibition
Effect on Lineweaver-Burk plot:
. Noncompetitive inhibition is readily differentiated from competitive inhibition by
plotting 1/vo versus 1/[S] and noting that the apparent Vmax decreases in the presence of a noncompetitive inhibitor, where as Km is unchanged.
33 Kinetics of Noncompetitive inhibition
34 Examples of noncompetitive inhibitors:
Lead forms covalent bonds with the sulfhydryl side chains of cysteine in proteins. Ferrochelatase, an enzyme that catalyzes the insertion of Fe2+ into protoporphyrin (a precursor of heme), is an example of an enzyme sensitive to inhibition by lead.
35 Non competitve inhibitor may be reversible Irreversible:
Reversible inhibitors Irreversible inhibitors
Attach non covalent Covalent tightly bound Can dissociate. to the enzyme.
36 Irreversible inhibitors
Irreversible inhibitor form permanent complexes with enzyme by make covalent bonds with enzyme. Inhibition cannot be reversed.
37 Irreversible inhibitors
Nerve gas, inhibitors such as DIFP, (diisopropyl fluorophosphates) was very potent and toxic inhibitors of acetylcholinesterase, an enzyme involved in nerve action. . If acetlycholine cannot be hydrolysed by this enzyme, nerve signals cannot be passed across the synapses of the nervous system. . On exposure to this compound, death can result in minutes due to respiratory failure.
38 39 3-Uncompetitive Inhibition
Uncompetitive Inhibitor binds only to ES complex at locations other than the catalytic Site, So formation of ESI alone. Substrate binding modifies enzyme structure, making inhibitor- binding site available. Inhibition cannot be reversed by substrate. In this case apparent Vmax and Km decreased.
40 3-Uncompetitive Inhibition
41 42 Kinetics of Uncompetitive Inhibition
43 Regulation of Enzyme Activity
44 Regulation of Enzyme Activity
The regulation of the reaction velocity of enzymes is essential if an organism is to coordinate its numerous metabolic processes.
Some enzymes with specialized regulatory functions respond to allosteric effectors or covalent modification, or they show altered rates of enzyme synthesis (or degradation) when physiologic conditions are changed.
45 A.Allosteric binding sites
Allosteric enzymes:
Enzymes have more than one site, where effector binding at one site induces a conformational change in enzyme altering its affinity for a substrate or modify the maximal catalytic activity of the enzyme.
46 A.Allosteric binding sites
Allosteric enzymes are regulated by molecules called effectors (also called modifiers) that bind noncovalently at a site other than the active site.
Effectors that inhibit enzyme activity are termed negative effectors, where as those that increase enzyme activity are called positive effectors.
47 Major features of allosteric enzymes
1) Multiple subunit .
2) Catalyze the committed step early in pathway.
3) Contain two seperate functional center: A)Catalytic B) Regulatory
4) May be subject to either +ve or –ve effectors
that affect either Vmax or Km or both. 48 Major features of allosteric enzymes
5)Effectors may be coenzymes, substrate or product.
6) Undergo changes either in conformation or in the cooperativity of componant polypeptides by appropriate effector.
7) Don’t follow the kinetics of Michaelis Menten model.
49 A.Allosteric binding sites
Effects of –ve or +ve effectors on an allosteric enzyme.
A. Vmax is altered.
B. The substrate concentration that gives half-maximal
velocity (K0.5) is altered
50 A.Allosteric binding sites
1-Homotropic effectors:
When the substrate itself serves as an effector. Most often, an allosteric substrate functions as a positive effector. In such a case, the presence of a substrate molecule at one site on the enzyme enhances the catalytic properties of the other substrate- binding sites that is, their binding sites exhibit cooperativity. • 51 A.Allosteric binding sites
2-Heterotropic effectors:
The effector may be different from the substrate(Feed back inhibition). The enzyme that converts D to E has an allosteric site that binds the endproduct G. If the concentration of G increases (for example, because it is not used as rapidly as it is synthesized), the first irreversible step unique to the pathway is typically inhibited. 52 2-Heterotropic effectors:
53 B.Regulation of enzymes by covalent modification 1-Phosphorylation & dephosphorylation: . Phosphorylation reactions are catalyzed by a family of enzymes called protein kinases that use ATP as a phosphate donor.
. Phosphate groups are cleaved from phosphorylated enzymes by the action of phosphoprotein phosphatases .
54 55 B.Regulation of enzymes by covalent modification
2-Response of enzyme to phosphorylation: • Depending on the specific enzyme, the phosphorylated form may be more or less active than the unphosphorylated enzyme. • Example . Phosphorylation of glycogen phosphorylase increases activity. . Phosohorylation of glycogen synthase decreases activity.
56 C.Induction and repression of enzyme synthesis
Cells can regulate the amount of enzyme present usually by altering the rate of enzyme synthesis.
The increase (induction) or decrease (repression) of enzyme synthesis leads to an alteration in the total population of active sites.
57 C.Induction and repression of enzyme synthesis
Enzymes subject to regulation of synthesis are often those that are needed at only one stage of development or under selected physiologic conditions.
For example, elevated levels of insulin as a result of high blood glucose levels cause an increase in the synthesis of key enzymes involved in glucose metabolism.
58 C.Induction and repression of enzyme synthesis Constitutive enzymes Inducible enzyme
Enzymes whose concentration of concentration remain enzyme depend on costant over the time. inducer may substrate or structurally related Due to>>>> rate of compound that initiate synthesis= rate of their synthesis. degradation.
59 C.Induction and repression of enzyme synthesis
Alterations in enzyme levels as a result of induction or repression of protein synthesis are slow (hours to days), compared with allosterically regulated changes in enzyme activity which occur in seconds to minutes.
60 D.Zymogen= Proenzyme or natural enzyme precursors Some enzymes are secreted in an inactive form called Proenzymes or zymogens.
At the site of action specific peptide bonds are hydrolysed either enzymatically or by PH changes to convert it into active form.
After hydrolysis when it is activated, it cannot be reconverted into proenzyme form.
61 D.Zymogen= Proenzyme or natural enzyme precursors
Digestive enzymes are released in inactive forms to prevent the digestive enzymes from autodigesting the cells that produce them.
Pancrease secretes zymogen to prevent pancreatitis to occur.
62 D.Zymogen= Proenzyme or natural enzyme precursors
• Examples:
Pepsinogen pepsin
Trypsinogen trypsin
Plasminogen plasmin
63 E.Feed back regulation
The 1st step is irreversile called feedback control.
If final product is accumulated, it will send single back to slow or inhibit the irreversible step.
64 E.Feed back regulation
End product may: 1) Act as repressor & block the synthesis of the enzyme. 2)-ve allosteric bind to senstive enzyme at allosteric site.
The kinetic of inhibition may be competitive or uncompetitive or noncompetitive allosteric regulation.
65 E.Feed back regulation
Cross regulation Asubstrate level control
Aproduct of one Direct interaction of S pathway serves as with the enzyme itself. inhibitor or activator of an enzyme occuring in another pathway.
66 Enzymes in Clinical Diagnosis & Therapy
67 Enzymes in Clinical Diagnosis
Plasma enzymes can be classified into two major groups: • First, a relatively small group of enzymes are actively secreted into the blood by certain cell types(the liver secretes zymogens (inactive precursors) of the enzymes involved in blood coagulation). • Second, a large number of enzyme species are released from cells during normal cell turnover.
68 Enzymes in Clinical Diagnosis
The presence of elevated enzyme activity in the plasma may indicate tissue damage that is accompanied by increased release of intracellular enzymes.
69 Alteration of plasma enzyme levels in disease states Many diseases that cause tissue damage result in an increased release of intracellular enzymes into the plasma.
Measurement of enzymes concentration in plasma gives valuable information about disease involving tissues of their origin.
70 1. Alkaline phosphates (ALP) conc.
In bone the enzyme is found in osteoblasts and is probably important for normal bone function.
The level of these enzymes may be increased in rickets and osteomalacia, hyperparathyroidism, obstructive jaundice, and metastatic carcinoma, congestive heart failure result of injury to the liver.
71 2. Transaminases Two transaminases are of clinical interest:
Aspartate transaminase Alanine transaminase (AST,GOT) (ALT,GPT) .It is widely distributed, with high .It is present in high concentration concentration, in the heart, liver, in liver and to a lesser extent in skeletal muscle, kidney and skeletal muscle, kidney and heart. erythrocytes, and damage to any of these tissues may cause raised levels.
.In myocardial infarction SGOT is .In liver damage, both SGOT & increased with little or no increase SGPT are increased, but SGPT in SGPT. increases more.
72 Isoenzymes and diseases of the heart Isozymes:
One of a group of enzymes that catalyze the same reaction but are differentiated by variations in physical properties due to difference in amino acid sequence, contain different number of charged amino acid.
73 1-Creatine kinase (CK) isozymes
CK enzymes consist of two subunits, which can be either B (brain type) or M (muscle type).
There are 3 different isoenzymes: 1. CPK-1 (CPK-BB) is found mostly in the brain and lungs. 2. CPK-2 (CPK-MB) is found mostly in the heart. 3. CPK-3 (CPK-MM) is found mostly in skeletal muscle .
74 75 1-Creatine kinase (CK) isozymes
Myocardial muscle is the only tissue that contains > 5% of the total CK activity as the CK2 (MB) isoenzyme.
Following an acute myocardial infarction, CK2 appears approximately 4 to 8 hours following onset of chest pain, reaches a peak of activity at approximately 24 hours, and returns to baseline after 48 to 72 hours.
76 2-Lactate dehydrogenase(LDH) It composed of 2 subnuits : 1. M(major form in muscle & liver) 2. H(major form in heart)
Can be combined into tetramers in 5 ways (M4, M3H, M2H2, MH3, H4).
LDH in heart can differentiated from LDH in liver by electrophoresis .
77 2-Lactate dehydrogenase(LDH)
Characters of LDH(H4) Characters of LDH(M4) Heart Liver -ve charged so migrate to +ve charged so migrate to anode cathod More resistant to heat Less resistant to heat Lactate to pyruvate(aerobic Pyruvate to lactate metabolism through CAC) (glycolysis under anaerobic condition) High affinity toward less affinity toward substrate substrate Inhibited by high level of Not inhibited by high level pyruvate of pyruvate 78 2-Lactate dehydrogenase(LDH)
79 Lactate dehydrogenase(LDH)
LDH activity is elevated in plasma following an infarction, peaking 2 to 3 days after the onset of symptoms.
80 Answer each of following
QI: Discuss in brief: • Lock and key theory of enzymes. • Allosteric enzyme features. • Regulation of enzymes by covalent modification. QII: Compare between each pair of the following: • Homotropic and heterotropic effectors. • Competitive and non competitive enzyme inhibitor. 81 82