Enzymes PDF

Faculty of Medicine 7 Medical Biochemistry I 0 Enzymes Dr. N. M. Elias  Enzymes Definition  Biological catalysts, protein catalysts or specialized proteins that accelerate the velocity of chemical reaction and are not...

Faculty of Medicine 7 Medical Biochemistry I 0 Enzymes Dr. N. M. Elias  Enzymes Definition  Biological catalysts, protein catalysts or specialized proteins that accelerate the velocity of chemical reaction and are not consumed or changed during the reaction they catalyze.  Almost all enzymes are proteins but not all proteins are enzymes.  Some RNA molecules, called ribozymes, can act as enzymes.  In the absence of enzymes, life, as we know it, could not exist.  100-300 AAs linked into unbranched polypeptide chains.  Substrate + Enzyme  Product + Enzyme  Enzymes nomenclature 1) Old system  Large number of enzymes were named according to their function.  Many of these enzymes catalyzed degradation reactions.  Simply named by addition of the suffix, -ase, to the name of the S. Substrate ase  For example, 1) Arginase, which hydrolyses Arg  urea + ornithine 2) Urease, which hydrolyses Urea  NH3 + CO2  Enzymes nomenclature  This practice was extended to non-degradative Es by the addition of -ase to a term descriptive of the catalyzed reaction to give, e.g., kinases, isomerases, oxidases, and synthetases. Substrate Reaction type ase  These terms were generally prefixed by the name of the S or, in the case of the synthetases, the name of the P. Product Synthetase  Enzymes nomenclature  Disadvantage has resulted in single enzyme possessing more than one name & in more than one enzyme bearing the same name. 2) New system  In 1961, IUBMB has overcome difficulties of old system by EC which proposed a 4-digits depending on the division of enzymes into 6 major classes according to the reaction they catalyze.  The 1st digit indicates classes. The next two indicate sub- and sub- sub-classes. The 4th gives the number of the particular E in the list. CLASSIFICATION OF ENZYMES  Classification of enzymes  Example IUBMB nomenclature EC: 1.1.1.1 = 1st digit: Class 1, oxidoreductase. 2nd digit: subclass, nature of the electron donor (CHOH group). 3rd digit: sub-subclass, nature of the electron acceptor (NAD+). 4th digit: sub-sub-subclass, order or name of the enzyme in such family (alcohol dehydrogenase), serial no. of the E. Alcohol + NAD+  aldehyde (or ketone) + NADH + H+  Structure of enzymes Apoenzyme  Refers to the protein portion of the holoenzyme. Nonrotein moiety  If it is a metal ion such as Zn2+ or Fe2+, it is called a cofactor.  If it is a small organic molecule, it is termed a coenzyme. Cofactors  Bind in a transient, dissociable manner either to the E or to a S e.g. inorganic molecule such as metal ions, Zn2+, Fe3+.  Structure of enzymes  Es that require a metal ion cofactor are metal-activated enzymes. Coenzymes  Function as group-transferring agents between metabolic pathways (e.g. NAD+, Co A & ATP) & do not function as part of the enzyme but fulfill the role of S (and P) since they are chemically modified at the end of a catalytic process.  Frequently derived from vitamins. For example, NAD + contains niacin and FAD contains riboflavin.  Structure of enzymes Cosubstrates coenzymes that only transiently associate with the E. Prosthetic group coenzyme is permanently associated with the E and returned to its original form (e.g. PLP, FMN, FAD, TPP, biotin & the metal ions of Co, Cu, Mg, Mn, Se, and Zn). Metal ions – tightly bound enzymes are called metalloenzymes. Holoenzyme (E+ cofactor)  In the absence of the appropriate cofactor, the apoenzyme typically does not show biologic activity.  Structure of enzymes Active site  3-D structure in the form of a cleft or pocket created as a result of Rs of the constituent AAs interactions. S + E  ES  EP  P + E  The interaction b/w E & Ss involves non-covalent bonds (in some cases covalent bonds are formed, but must be sufficiently weak).  Acts as a flexible molecular template that binds the S and initiates its conversion to the T.  Structure of enzymes Isoenzymes (or isozymes or isoforms)  They often differ from each other by only a few AA residues (primary structure encoded by different genes) and may have very similar M wts (different physical, kinetic, or regulatory properties).  May occur in the same cell in different subcellular sites (e.g. cytosol & mitochondria) or in different organs (e.g. liver & muscles). Ex LDH has 5 isoenzymes, H4, H3M, H2M2, HM3 and M4. HK has 4 isoenzymes, I, II, III, and IV. CK has 3 isoenzymes, CK1 (BB), CK 2 (MB) & CK3 (MM).  Structure of enzymes Zymogen (proenzyme) Proteolytic activation  A mechanism for activation of proteins outside cells.  Several Es are synthesized as larger inactive precursor forms.  Involves irreversible hydrolysis of one or more peptide bonds. Examples o Gastric & pancreatic proteases (hydrolyze dietary proteins) Synthesized in the stomach and pancreas as zymogens.  Structure of enzymes Only upon proteolytic activation, form an active S-binding site. Pancreatic and gastric zymogens Origin Zymogen Active protease Pancreas Trypsinogen Trypsin Pancreas Chymotrypsinogen Chymotrypsin Pancreas Procarboxypeptidase Carboxypeptidase Pancreas Proelastase Elastase Stomach Pepsinogen Pepsin  Structure of enzymes o Blood clotting cascade The formation of blood clots results of a series of zymogen activations. Seven of the clotting factors in their active form are Ser proteases: kallikrein, XIIa, XIa, IXa, VIIa, Xa, and thrombin. o Insulin An important metabolic regulator, is generated by proteolytic excision of a specific peptide from proinsulin.  Proteolytic removal of residues 31 to 65 yields insulin. Residues 1 - 30 (the B chain) remain linked to residues 66 - 86 (the A chain) by a pair of interchain disulfide bridges.  Properties of enzymes 1) High catalytic activity:  Enzyme – catalyzed reactions proceeding from 103 to 108 times faster than unanalyzed reactions.  Typically each E is capable of transforming 100 to 1000 S  P/Sec.  The number of S  P/E /Sec is called the turnover number. 2) Role of AAs:  Only a small proportion of the AAs present in a protein are involved directly in the active site.  Properties of enzymes  Other AAs may play a role in: A. Maintenance of the 3-D structure of the enzyme. B. Attaching the enzyme to IC structures (e.g. membranes). C. Binding molecules that regulate the activity of the enzyme. 3) Specificity:  Formation of the ES complex occurs only if S possesses groups which interact with the binding groups of the active site (lock and key model).  Properties of enzymes  Ranging from absolute (e.g. GK) to group specificity (e.g. HK).  The vast majority of enzymes exhibit stereospecificity; thus HK phosphorylates D-glucose but not L-glucose. 4) Induced fit:  The binding of the correct S triggers a change in the structure of the E (i.e. conformation change) that brings catalytic groups into exactly the right position to further  the specificity.  Properties of enzymes  The lock & key model for E specificity uses complementarity b/w the E active site (the lock) and the S (the key).  Simply, the S must fit correctly into the active site—it must be Right size and shape, Have charges in the correct place, Have the right H-bond donors & acceptors, Have just the right hydrophobic patches. Properties of enzymes  Properties of enzymes 5) Denaturation:  A number of factors destroy E activity as a consequence of their ability to denature proteins (disturbance of the 3-D structure).  Denaturing agents include heat, extremes of PH, organic solvents, detergents and  [urea].  Agents are able to disrupt the non-covalent forces b/w AA residues in the E that determine its overall 3-D structure.  Distribution in the cell  In general, Es are synthesized in the cell cytoplasm and are then exported to the place where they fulfill their mission.  Es that are secreted act outside the cell (digestive Es or CFs).  The vast majority of enzymes are IC. Associated with the nucleus (maintenance & function of the genes). In mitochondria (oxidative reactions to supply energy). In ribosomes (protein synthesis).  Distribution in the cell In lysosomes, hydrolases degrade molecules at the end of their life at a pH that is more acidic than that of other cell enzymes. In microsomal fractions (synthesis of complex lipids & in metabolism & inactivation of foreign substances). In Golgi complex (synthesis of oligosaccharides & the glycosylation of proteins & lipids). In cytosol (glycolysis & biosynthesis of FAs & other compounds). In plasma membrane (transport of solutes).  Enzyme kinetics The transition state (activated complex or half-way house)  A -energy intermediate is formed during conversion of R  P.  All chemical reactions have an energy barrier separating the reactants and the Ps.  This barrier, called the free energy of activation, is the energy difference b/w reactants and a high-energy intermediate.  In E reactions, while stable arrangement of atoms (S)  another (P), the atoms will pass through unstable arrangement.  Enzyme kinetics  The effect of any catalyst is to lower the activation energy.   activation energy, the rates of uncatalyzed reactions are slow. Rate of reaction  For molecules to react, they must contain sufficient energy to overcome the energy barrier of T.   the free energy of activation,  molecules have sufficient energy to pass through T (faster the rate).  Chemical kinetics  Simplest possible reaction, the irreversible conversion of A to B K1 A B  The rate of P formation contains a rate constant (k).  Reaction rate, or velocity (v), at any instant is the rate of formation of the P or consumption of the S. ( [B] or  [A] with ∆ [ ] ∆ [ ] time): = or =− ∆ ∆  v is the number of S molecules converted to P per unit time and is usually expressed as μ moles P formed/min.  Chemical kinetics  The K provides a direct measure of how fast this reaction is. The larger the value of k1, the more rapid the rate and vice versa.  The order of a reaction is determined experimentally by comparing the kinetic data (i.e., a plot [A] vs. time). Uncatalyzed reactions, S reactions follow first-order kinetics. First-order reaction, the V is directly proportional to the [S]. Zero-order kinetics are observed only in catalyzed reactions when the [S] is . A zero-order reaction is independent of the [S]. Chemical kinetics  Factors affecting reaction rate 1) [S]  The rate of an enzyme-catalyzed reaction increases with [S] until a maximal velocity (Vmax) is reached. The lower region of the curve, the rxn approaches 1st-order kinetics, i.e. v is a direct function of [S] because unsaturated active sites. At the plateau, the reaction approaches zero-order kinetics because the active sites of all the Es are saturated & the reaction rate is independent of further  in [S].  Factors affecting reaction rate The intermediate portion of the curve, as the E approaches S saturation, kinetics are mixed 0 & 1st order in [S].  Most Es show Michaelis-Menten kinetics, and a plot V∘ & [S], has a hyperbolic shape similar to the ODC of Mb. In contrast, allosteric enzymes frequently show a sigmoidal curve.  Factors affecting reaction rate 2) Temperature  The reaction rate  with  temp until a peak velocity is reached.  This  is due to the  number of molecules having sufficient energy to pass over the energy barrier and form P of the reaction.  Further  of the temp   in reaction velocity (denaturation).  The optimum temp. range for most Es varies b/w 30 and 45°C.  Most enzymes are very rapidly denatured at 100 °C and show a  activity after quite short exposures to temperature above 60°C.  Factors affecting reaction rate   temp.s to near or below 0°C although inactivate the E but this is a reversible type of change and the E regains its catalyzing power upon  the temperature to optimum.  Factors affecting reaction rate 3) [H+]  Changes in PH will modify the degree of ionization of some or all of ionizable groups and this will affect the ionization of the E as a whole. For example, catalytic activity may require that an amino group of the E be in the protonated form (–NN3+ ). At alkaline pH this group is deprotonated & the reaction rate . 1) Extremes of pH (< 4.0 or > 10.0) will change the degree of ionization of many groups in the enzyme causing denaturation.  Factors affecting reaction rate 2) Smaller changes of pH (4 to 10) will affect a smaller number of ionizable groups but if these happen to be present in the active site they will cause a marked change in E activity. This effect is completely reversible. 3) Changes in pH may also cause changes in the ionization of S and this could modify the binding of S to E.  Some Es are clearly adapted to function at the particular PH of their normal environment.  Factors affecting reaction rate 4) [E]  The rate of an enzymatic reaction is always directly dependent on [E].  The higher enzyme level, the faster reaction velocity will proceed, and therefore the relationship between [E] & reaction velocity is linear.  Michaelis – Menten equation  Michaelis and Menten proposed a simple model for E catalysis K1 K3 E+S ES P+E K2 Where: K1,K2, and K3 are rate constants  The Michaelis-Menten equation describes how v varies with [S]: Vmax [S] V∘ = Km + [S] Km = Michaelis constant = (K2 + K3)/K1 Km  Equal to the sum of rates of breakdown of ES/rate of formation.  Michaelis – Menten equation  Numerically equal to the [S] at which the v is equal to ½ Vmax.  Km does not vary with the [E].  It reflects the affinity of the E for that S. Numerically ↓ Km (K1 predominant over K2)  strong S binding (high affinity of the E for S) because a ↓ [S] is needed to ½-saturate the E that is, reach a velocity that is ½ Vmax. Numerically ↑ Km (K2 predominant over K1) weak S binding, (low affinity of E for S) because ↑ [S] is needed to ½-saturate the E.  Michaelis – Menten equation  When [S] < Km, Km + [S] is essentially equal to Km [ ] [ ] ∘ = ≅ ≅ [ ] + [ ]  Vmax and Km are both constants, their ratio is a constant. V∘ is directly proportionate to [S]  When [S] > Km, Km + [S] is essentially equal to [S]. [ ] [ ] ∘ = ≅ ≅ +[ ] [ ] The reaction velocity is maximal & unaffected by further  in [S]. [ ] [ ]  When [S] = Km. ∘ = ≅ ≅ +[ ] [ ]  Michaelis – Menten equation Order of reaction: When [S] is much less than Km, the velocity of the reaction is ~ proportional to the [S]. The rate of reaction is then said to be first order with respect to S. When [S] is much greater than Km, the velocity is constant and equal to Vmax. The rate of reaction is independent of [S], and is said to be zero order with respect to [S].  Lineweaver-Burke plot  Used to calculate Km and Vmax, as well as to determine the mechanism of action of enzyme inhibitors.  When V∘ is plotted against [S], it is not possible to determine Vmax, because of the gradual upward slope of the hyperbolic curve at  [S] but if 1/V∘ is plotted versus 1/[S], a straight line is obtained.  Lineweaver-Burke plot  This plot is a derivation of the Michaelis–Menten equation: = + ∘ [ ]  A Linear form of the Michaelis-Menten equation, with the intercept on the Y-axis equal to 1/Vmax, and the intercept on the X-axis equal to -1/Km. The slope of the line is equal to Km/Vmax.  A disadvantage of the Lineweaver–Burk plots is that for small values of [S], small errors in V∘ lead to large errors in 1/ V∘ and hence to large errors in KM & Vmax.  Inhibition of enzyme activity  Inhibitor is any substance that can diminish or stop catalysis.  They may be small inorganic ions, or organic substances.  Some E inhibitors are normal body metabolites that inhibit a particular E as part of the normal metabolic control of a pathway.  Other inhibitors may be foreign substances, such as drugs or toxins, where the effect of E inhibition could be either therapeutic or, at the other extreme, lethal.  The reduction in E activity can be irreversible or reversible.  Inhibition of enzyme activity Irreversible inhibitors o Bind tightly, often covalently, to AA residues at the active site of the E, permanently inactivating the E. The inhibited E does not regain activity upon dilution of the enzyme-inhibitor complex. o These inhibitors are usually toxic substances that poison enzymes. o Examples, 1) Diisopropylfluorophosphate (DIFP, insecticide)  Inhibition of enzyme activity A component of nerve gases, reacts with a Ser residue in active sites of Es, e.g. (1) Digestive Es (trypsin, chymotrypsin; proteases), (2) Ach-esterase (deactivate the transmission of nerve impulses). Resulting in paralysis of vital body functions. 2) Iodoacetamide & iodoacetate reacts with -SH at the active site. Iodoacetate is an inhibitor of the Es like papain & G3PD. 3) Cyanide inhibits cytochrome oxidase (binds to iron atom) of ETC. 4) Fluoride inhibits enolase (by removing Mn), and thus glycolysis.  Inhibition of enzyme activity 5) Disulfiram (Antabuse®) a drug used in treatment of alcoholism. It irreversibly inhibits aldehyde dehydrogenase. Alcohol addicts, when treated with disulfiram become sick due to  acetaldehyde  alcohol avoidance. (Alcohol  alcohol DH  acetaldehyde  aldehyde DH  acetic acid.) 6) Suicide inhibition The original inhibitor (structural analogue/competitive)  more potent form by the same E.  Inhibition of enzyme activity The Ps establish covalent bonds that irreversibly block the active site of the E, as if the E had “committed suicide.” A good example is allopurinol (used in treatment of gout). Allopurinol, an inhibitor of xanthine oxidase, gets converted to alloxanthine, a more effective inhibitor of XO. Use of certain purine & pyrimidine analogues in cancer therapy, e.g.,5-fluorouracil  fluorodeoxyuridylate  inhibits thymidylate synthase  disrupt DNA synthesis in rapidly dividing cancer cells.  Inhibition of enzyme activity Aspirin is the most commonly used drug for relieving pain and as anti-inflammatory. Aspirin acetylates Ser in the active center of COX thus inhibiting the PG synthesis and the inflammation. Enzyme inhibition by drugs  Many of the drugs act as enzyme inhibitors. For example : 1) Statin (lovastatin) inhibits HMG CoA reductase (cholesterol). 2) Tenofovir & emtricitabine inhibit viral reverse transcriptase (employed to block HIV replication).  Inhibition of enzyme activity 3) Omeprazole, to treat gastroesophageal reflux disease, irreversibly inhibits the proton pump in the stomach   [acid] in the stomach and the associated “heartburn.” 4) Captopril & enalapril inhibit ACE (treat hypertension). 5) Penicillin covalently attaches to Ser in the active site of glycopeptides transpeptidase (forms the cross-links in the bacterial cell wall), i.e. irreversible inhibitions  Inhibition of enzyme activity Reversible inhibitors  Bind to Es through noncovalent bonds and can always be reversed by removal of the inhibitor.  Dilution of the E-I complex results in dissociation of the reversibly- bound inhibitor and recovery of enzyme activity.  In some cases, noncovalent binding may be so strong as to appear irreversible under physiological conditions, e.g. trypsin inhibitor.  They are competitive or noncompetitive.  Inhibition of enzyme activity 1) Competitive inhibition  Inhibitor typically has close structural analogue to the normal S.  Thus, it binds reversibly to the active site and competes with the S for that site but does not undergo any catalysis.  Inhibition of enzyme activity  Both the ES and EI complexes are formed during the reaction.  So the affinity of the S for the E is progressively  with the  in [I] lowering the rate of enzymatic reaction.  When [S] , the effect of inhibitor can be reversed forcing it out. 1. Effect on Vmax: The effect is reversed by  [S]. At a sufficiently  [S], the V reaches the Vmax observed in the absence of I. 2. Effect on Km: A competitive inhibitor  Km, i.e. presence of a competitive inhibitor more S is needed to achieve ½ Vmax.  Inhibition of enzyme activity 3) Example: o SDH catalyzes the oxidation of succinate to fumarate. Malonate is structurally similar to S and competes for binding  E-malonate complex is unreactive. By  [succinate]  active site is occupied by S instead of I. o HTN can be treated with inhibitors of ACE (metalloprotease). Captopril is a structural analog of angiotensin I, which is a short peptide: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe—His-Leu.  Inhibition of enzyme activity Cleavage of the Phe—His peptide bond angiotensin II, which causes  BP. Captopril binds tightly to several Rs in the ACE active site, as well as the Zn2+ ion that is required for activity  blocks the binding of angiotensin I to ACE active site. Captopril binds ACE ∼ 25,000 times more tightly than does angiotensin I. Statins (antihyperlipidemic agents) competitively inhibit HMG- CoA reductase. By doing so, they inhibit de novo cholesterol synthesis, and lowering plasma cholesterol levels.  Inhibition of enzyme activity o Allopurinol: UA is formed in the body by oxidation of hypoxanthine by XO. Allopurinol structurally resembles hypoxanthine and inhibits XO   UA formation.  Inhibition of enzyme activity o Sulphonamides: antibacterial agent. ρ-aminobenzoic acid (PABA) is essential for synthesis of folic acid which is needed for bacterial growth & survival. Bacterial wall is impermeable to FA. Sulphonamides are structurally similar to PABA and inhibit the E. Thus, FA is not synthesized & bacterial growth suffers & they die. o MAO inhibitors: MAO oxidizes catecholamines. Ephedrine & amphetamine competitively inhibit MAO & prolong the action of pressor amines.  Inhibition of enzyme activity o Methotrexate: chemically it is 4-amino-N 10 -methyl FA. It structurally resembles FA & competitively inhibits “folate reductase” (essential for DNA synthesis & cell division) and prevents formation of FH4. Hence, DNA synthesis suffers. Therefore, methotrexate is used as an anticancer drug. o Physostigmine: a drug which competitively inhibits Ach-esterase (hydrolyses Ach  choline & acetate) & prevents destruction of Ach  continued effect of Ach in post-synaptic region.  Drug Enzyme inhibited Clinical use Allopurinol Xanthine oxidase Gout Dicoumarol Vitamin K-epoxide- reductase Anticoagulant Penicillin Transpeptidase Antibiotic Sulfonamide Pteroid synthetase Antibiotic Trimethoprim FH2-reductase Antibiotic Pyrimethamine Do Malaria Methotrexate Do Cancer 6-mercaptopurine Adenylosuccinate synthetase Cancer 5-fluorouracil Thymidylate synthase Cancer Azaserine Phosphoribosyl-amidotransferase Cancer Cytosine arabinoside DNA polymerase Cancer Acyclovir Do Virus Neostigmine ACh-esterase Myasthenia Alpha-methyl dopa Dopa-decarboxylase Hypertension Lovastatin HMGCoA-reductase Cholesterol lowering Oseltamiver (Tamiflu) Neuraminidase Influenza  Inhibition of enzyme activity o Dicoumarol: used as an anticoagulant. It is structurally similar to vitamin K and competitively inhibiting it. o Succinylcholine: a muscle relaxant structurally similar to Ach. It competitively fixes on post-synaptic Rs. Not hydrolyzed by Ach- esterase  continued depolarization  muscle relaxation. 2) Non-competitive inhibition  Binds at a site other than the active site  I combine with both free E & ES complexchange in 3-D shape   catalytic activity.  Inhibition of enzyme activity  This is of two different types: (i) reversible and (ii) irreversible.  If the I can be removed without affecting the activity of the E, it is called reversible-non-competitive inhibition but If it leads to loss of E activity, it is known as irreversible non-competitive inhibition.  Inhibition of enzyme activity  Since the I binds at a different site to the S, the E may bind the inhibitor, the S or both the I and S.  In many reversible inhibitors both the E and the ES bind I. 1. Effect on Vmax: Noncompetitive inhibition cannot be overcome by  [S]. Noncompetitive inhibitors  the Vmax of the reaction. 2. Effect on Km: Noncompetitive inhibitors do not interfere with the binding of S to E. Thus, the E shows the same Km in the presence or absence of the noncompetitive inhibitor.  Inhibition of enzyme activity 4) Example: o Lead forms covalent bonds with SH of Cys in proteins. Ferrochelatase & δ-ALA dehydrase are inhibited by lead. Uncompetitive inhibition  It binds only to ES complexes at locations other than the catalytic site. S binding modifies E structure, making inhibitor- binding site available. Inhibition cannot be reversed by S.  Inhibition of enzyme activity 1. Effect on Vmax: Since it cannot be overcome by  [S]. Thus, uncompetitive inhibitors  the Vmax of the reaction. 2. Effect on Km: is .  Competitive Noncompetitive Uncompetitive Binding site Active site Other than active site Other than active site Binding to E Free E Free E or ES ES Effect on Vmax No   Effect on Km  No  Reversibility Yes Yes or No No  Regulation of enzyme activity  The enzyme activity in cells changes constantly and it is adjusted to physiological requirements.  Es must be controlled in a manner that reflects the availability of Ss, the utilization of products, and the overall needs of the cell.  E activity is directly proportional to the level of S. The [S] is  or close to the E Km, i.e. the [S] determines the degree of E activity.   the [S] in the cell accelerates its utilization and vice versa.  Enzyme activity can be regulated in several ways:  Regulation of enzyme activity 1) Allosteric regulation. 2) Covalent modification, or 3) Enzyme synthesis and breakdown. Allosteric binding sites Allosteric Es are regulated by molecules (effectors, modifiers or modulators) bind non-covalently at a site other than the active site. The presence of an allosteric effector can alter the affinity of the E for its S or modify the maximal catalytic activity of the E or both.  Regulation of enzyme activity Effectors  E activity (-ve effectors or allosteric inhibitor), or  E activity (+ve effectors or allosteric activator). Allosteric Es usually contain multiple subunits and frequently catalyze the committed step early in a pathway & is feedback- inhibited by the end-product of the pathway. Substrate-level control:  [S], the more rapidly a reaction occurs, at least until saturation of the E is approached. Conversely,  [P], which can also bind to the E, tend to inhibit the S to P.  Regulation of enzyme activity Ex, HK, is inhibited by its P, G6P. If subsequent steps are blocked, G6P will  and bind to HK P inhibition   production of G6P. In many cases the P binds the active site & acts as a competitive I. 1. Homotropic effectors:  When the S itself serves as an effector, the effect is homotropic.  Most often an allosteric S functions as a positive effector.  In such a case the presence of S at one site on the E enhances the catalytic properties of the other S-binding sites– cooperativity, Hb.  Regulation of enzyme activity  These enzymes show a sigmoidal curve when Vo is plotted vs [S].  This contrasts with the hyperbolic curve following M-M kinetics.  +ve & -ve effectors can affect either the Vmax, the K0.5, or both.  Regulation of enzyme activity 2. Heterotropic effectors:  The effector may be different from the S, e.g. the enzyme that converts A to B has an allosteric site that binds the end-product E.  Regulation of enzyme activity  If the [product E]  (because it is not utilized as rapidly as it is synthesized), the initial E in the pathway is inhibited.  Feedback inhibition provides the cell with appropriate amounts of a P it needs by regulating the flow of S molecules through the pathway that synthesizes that product.  Heterotropic effectors are commonly encountered, e.g. PFK-1 is allosterically inhibited by citrate, which is not a S for the enzyme.  Regulation of enzyme activity Covalent modification (phosphorylation/dephosphorylation)  The majority of enzymes, and their associated metabolic and signaling pathways, are regulated by reversible phosphorylation.  Protein kinases are ATP-dependent Es that add a phosphoryl group to the -OH of Tyr, Ser, or Thr on some target protein.  This process is made reversible by phosphatases.  Other types of reversible covalent modification used to regulate certain Es include adenylylation, ADP-ribosylation and acetylation.  Regulation of enzyme activity  Examples of Es activated by phosphorylation: Glycogen phosphorylase. HMG-CoA reductase kinase. F1,6BPase & F2,6BPase  Es activated by dephosphorylation: e.g. Glycogen synthase. PFK-2. Pyruvate kinase (PK) & Pyruvate dehydrogenase(PDH).  Regulation of enzyme activity Induction and repression of enzyme synthesis  The regulatory mechanisms described previously modify the activity of existing enzyme molecules.  Cells can also regulate the amount of E present (by altering the rate of E synthesis).  The amount of E present is a balance b/w the rates of its synthesis and degradation.  Regulation of enzyme activity  The  (induction) or  (repression) synthesis of the protein leads to an alteration in the total population of active sites, rather than influencing the efficiency of existing E molecules.  Most enzymes that are important in metabolic regulation have short half-lives, and are termed labile enzymes.  Enzymes whose concentrations remain essentially constant over time are termed constitutive enzymes.  Regulation of enzyme activity Time required for Regulator event Typical effector Results change Substrate availability Substrate Change in velocity Immediate Product inhibition Product Change in Vm & or Km Immediate Immediate Allosteric control End product Change in Vm & or Km (sec-mins) Immediate to Covalent modification Another enzyme Change in Vm & or Km minutes Synthesis or Hormone or Change in the amount of Hours to days degradation metabolite the enzyme  Clinical applications of enzymes  Enzymes in blood can be divided into 2 categories: 1) Normally present and have a specific role in body fluid (e.g. Es of the blood clotting mechanism; cholineslerase). 2) Released into the bloodstream from damaged tissues.  Enzymes as sites for drug action  Enzymes as laboratory reagents.  Differential diagnosis.  Clinical applications of enzymes Serum elevation of enzyme Disease Amylase Acute pancreatitis ACP Prostatic carcimona Liver disease specially biliary obstruction & detection of ALP osteoblastic bone disease e.g. rickets AST Myocardial infarction & liver disease ALT Liver disease specially liver cell damage LDH Myocardial infarction & liver disease Myocardial infarction & skeletal muscle disease (muscular CK dystrophy, dermatomyositis) GGT Liver disease, particularly obstruction & alcoholism Urinary elevation of N-acetyl Renal transplant rejection glucosaminidase  Clinical applications of enzymes  Monitoring therapy Measurement of the activities of Es in the blood can be used to assess therapy & indeed to control the dose of therapeutic agents given to the patients (e.g. AST & ALT activity after steroid or immunosuppressive therapy for active chronic hepatitis).