Biochemistry-Block-2.1-Enzymes.pdf

Biochemistry | BLOCK 2 Enzyme (including enzyme kinetics) Dr. Celina Vilches | 9/8/2022 | 9:30-11:30 AM Transcribed by: Pedro, Rodriguez, Samaniego, Samillano...

Biochemistry | BLOCK 2 Enzyme (including enzyme kinetics) Dr. Celina Vilches | 9/8/2022 | 9:30-11:30 AM Transcribed by: Pedro, Rodriguez, Samaniego, Samillano ▪ By breaking old covalent bonds OUTLINE and forming new covalent I. Enzymes bonds A. Definition B. Properties Properties of Enzymes C. Classification Effective and Efficient Catalyst i. Based on the catalyzed reaction - Rate of reaction catalyzed by an enzyme ii. Based on composition is much more rapid than the rate of the D. Non-Protein Molecule same reaction taking place in the II. Prosthetic Group absence of the enzyme. i. Cofactor carbonic anhydrase ii. Coenzyme Ex. CO2 + H2O Carbonic Acid (100 sec) E. Active Site 1M molecule of carbonic acid/sec F. Lock & Key Model G. Induced Fit Model - Increases the rate of non catalyzed H. Enzyme – Substrate Complex reaction by at least 106 (Lippincott: 103 I. Changing the Active Site to 108) J. Mechanisms Facilitating Catalysis Highly specific catalysts i. Catalysis by Proximity - Specific as to type of reaction they ii. Acid – Base Catalysis catalyzed (Reaction Specific) iii. Covalent Catalysis - Acts on single substrate or a small set of iv. Catalysis by Strain closely related substances K. Catalytic Residues (Substance/Substrate Specific) L. Isoenzymes - Typically catalyzed only one M. Diagnostic Values of Enzymes stereoisomer of a given compound N. Detection of Enzymes (Stereospecific) O. Enzyme Assays Protein catalysts and contain active site/s i. ELISA - Increases the velocity of a chemical ii. Spectrophotometric Assay reaction and are note consumed during iii. Coupling Dehydrogenase the reaction they catalyze. P. Diagnosis of Genetic Disease - All enzymes are proteins except for the Q. Tools for Studying Enzymes catalytic RNAs, the ribozymes which is a nucleic acid. ENZYMES General Characteristics Definition Nomenclature: I. Biological catalyst Commonly used name: o Have complex structure (sequence of amino o Typically named by the reaction they catalyze acids) followed by the suffix -ase. Example: Oxidase, o Act only upon a specific substrate Proteases o Do not change direction (energetics) of reaction o Modifiers can precede the name and may o Convert substrates to products without indicate: changing themselves ▪ Substrate (Xanthine Oxidase) o Mostly protein ▪ Source of the enzyme (Salivary Amylase) That accelerate or catalyze chemical ▪ Its regulation (Hormone sensitive Lipase) reactions (A to B) in cells ▪ Feature of its mechanism of action (Cystiene protease) Systemic Name: Catalyze cleavage of bonds by addition of According to the IUMB water Six major classes of enzymes Hydrolase, Protease, Lipase Based on Composition: Simple enzymes - Composed wholly of protein Complex enzymes - Composed of the protein + a relatively small organic molecule - Holoenzymes ▪ Apoenzymes − protein component ▪ Coenzyme/prosthetic group/co- factor − small organic molecule − Participate directly in substrate binding or Nomenclature by the IUMB catalysis 4-digit numbering system − increases the catalytic o 1st # - one of the 6 major classes of enzyme capacity activity o 2nd # - subclass The non-protein molecules/small organic molecules ▪ Type of bond acted upon or type of can be coenzymes, prosthetic groups or cofactors reaction it catalyzes which are derivatives of Vit. B or metal ions and extend o 3rd # - subsubclass the catalytic capabilities of the enzymes. ▪ Group acted upon, cofactor required, etc. Non-Protein Molecules o 4th # - serial number Prosthetic Groups Tight, stable association incorporated into protein structures of the enzyme by covalent or Classification noncovalent forces Based on the Catalyzed Reaction: Examples: Types of Chemical Reactions: Pyridoxal phosphate Oxidation Flavin Mononucleotide (FMN) Loss of electrons Flavin Adenine Nucleotide (FAD Transfer of electrons from donor to oxygen Metal ions – Co, Cu Oxidase, dehydrogenase o Metals are the most common prosthetic Reduction and co-factor group Gains electrons o Fe, Co, Cu, Mg, Mn, and Zn Reductases Metalloenzymes Transferases participates in redox reactions Catalyze transfer of C, N or P-containing complexed to prosthetic groups such as groups heme o Exchange reaction Action of metals o Addition Facilitate the binding and orientation of o Subtraction substrates Kinase, Transaminase, Transferase, Facilitate the formation of covalent bonds with Phosphorylase, Aminase, Phosphatase, reaction intermediates Deaminase Interacts with substrate to render them more Hydrolase electrophilic or nucleophilic  Isozymes Cofactors Enzymes belonging to the same family and Bind in a transient, dissociable manner either to employs a similar mechanism to catalyze a the enzyme or to substrate such as ATP (cofactor- common reaction but acts on different substrate substrate complex) Catalyze the same reaction but differences in Must be present in the medium surrounding the properties may be in: enzyme for catalysis to occur o Sensitive to a regulatory factor Metal ions are the cost common cofactor o Substrate affinity Metal ions: Cu, Mg, Mn, Fe which act as Importance: activators &/or inhibitors of enzyme activity o Adapt to certain tissue or circumstances Metal activated enzymes for diagnostic purposes o Enzymes that require metals as a o Enhance survival by providing a “back-up cofactor copy” ▪ Common ancestry (homologous) Coenzymes − Products of genes that Serves as recyclable shuttles or group transfer vary only slightly agents on substrates − Presence of specific o Transport many substrates from their amino acid ions on the point of generation to their point of same position in each generation to the point of utilization family member Coenzyme Q in the ETC stabilizes substrates such as (Conserved residues) hydrogen atoms or hydride ions that are unstable in *Catalytic residues aqueous environment of the cell Enzyme belonging to the same family employs a Chemical moieties transported by the coenzymes similar mechanism to catalyze a common o Methyl group (Folates), reaction but acts on a different substrate o Acyl Groups (Coenzyme A) - Gene duplication (2nd copy) → each copy o Oligosaccharides (Dolichol) developed independently → ability to perform the same reaction but using the Based on Substrate Type different substrate Specific for the kind of reaction they catalyze Example: Dehydrogenases – redox reaction Chemotrypsin - Cleaves peptide bonds on the Specific for the kind of substrate carboxyl terminal of large hydrophobic amino o Single substrate acids Succinate dehydrogenase – Trypsin - Cleaves peptide bonds on the carboxyl succinic acid terminal of basic amino acids o Broad spectrum − Most active against one Active Sites particular substrate Special pocket or cleft on the enzyme surface Alcohol dehydrogenase Multimeric enzymes – interphase (clefts) – different alcohols between subunits (ethanol) Contains amino acid side chains that create a 3D Specific to a particular steric configuration (optic surface complementary to the substrate isomer) of a substrate Results a high substrate specificity and catalytic ▪ Racemases efficiency − convert D isomers to L Many amnioacyl residues constitute the active isomers and vice versa site Substrate can be acted upon by a number of Binds with the substrate at the binding site, specific enzymes forming the enzyme substrate complex -> EP o Uses same substrates and produces the which subsequently dissociates to enzyme and same products product ▪ Isozymes  Folds to precisely fit the contours of a substrate via weak electrostatic interactions & facilitates bond reactivity o A cleft or a pocket in the structure of the enzyme that creates an environment exquisitely tailored for a single reaction. ▪ Results a high substrate specificity ▪ Results a high catalytic efficiency For a reaction to occur: Molecules should come within bond forming Acid-base catalysis distance to one another Ionizable functional groups of aminoacyl Bonds are broken and there is formation of new side chain and of the prosthetic group bonds can contribute to the catalysis by acting Enzyme substrate complex as an acid or a base Unique joining of enzyme and substrate at active Catalysis by strain site Mechanism used by enzymes that Functions of the ES complex: catalyze (break bonds aka lytic reactions) holds substrate out of aqueous solution ▪ Results in cleavage or breaking down holds substrate in specific orientation, ▪ Binds their substrate in a close to transition state to allow reaction conformation slightly unfavorable to occur for the bond that will undergo reduces ability of free rotation & cleavage → cause a strain on the molecular collisions with non-reactive bond → stretch or distort the bond atom → weakens the bond making it more allows amino acid side chains to alter vulnerable to cleavage local environment changes ionic strength, pH, adds or Covalent catalysis removes H-bonds to substrate Involves the formation of covalent bond between the enzymes and one or more Enzyme substrate interaction substrate 1. Catalysis by proximity and orientation Example: 2. Catalysis by bond strain (catalysis by strain) 3. Catalysis involving proton donors (acid) and Enzyme + substrate becomes a reactant acceptors (base) aka Acid-Base catalysis A+B➔C 4. Covalent catalysis A + B + E ➔ A-E-B ➔ C + E (lower activation energy therefore faster reaction) Mechanisms to Facilitate Catalysis Catalysis by Proximity and Orientation The higher the substrate concentration Enzyme substrate interactions the faster will be the chemical reaction Lock and Key Model Attachment of substrate to the active site Proposed by Emil Fischer in 1894. ▪ Will create a region of high local “Union between substrate – enzyme” theory substrate concentration Active site is presumed to be pre-shaped to fit the ▪ Proper orientation or ideal position substrate for a chemical rection to occur There is no change in the active site before and after a chemical reaction. visualize the interactions between the substrate and the enzyme in terms of the “lock and key”  ELISA (Enzyme Linked Immunosorbent Assay) Uses antibody covalently linked to a reporter enzyme whose products are readily detected via light absorbance or by fluorescence. Induced Fit Model Proposed by Danial Kosh Land in 1958. Describes that only the proper substrate is capable of inducing the proper alignment of the active site that will enable the enzyme to perform its catalytic function. Enzyme-Substrate causes a change in the shape of Spectrophotometric Assay the active site and alteration of chemical bonds. Exploits the ability of a substrate or product to The initial interaction between enzyme and absorb light substrate is relatively weak. Examples Then rapidly induce conformational changes in the Reduced NADH and NADPH 􀀀 absorb light at 340 enzyme that strengthen binding and bring catalytic mm sites close to substrate bonds to be altered. Oxidized form of NAD & NADP do not After binding takes place, one or more mechanisms NAD(P) is reduced - increased absorbance of of catalysis generates transition-state complexes and light reaction products. NAD(P) oxidized decreased absorbance Rate of change in the optical density at340nm will be proportionate to the degree of the of the enzyme present Coupling Dehydrogenase The activity of the enzyme can be determined by measuring the remaining substrate or the amount of product present. Separation of the product and the substrate Use a radioactive substance By coupling with a dehydrogenase Detection of enzymes Serum Enzymes used in Clinical Diagnosis Single-Molecule Enzymology Is based on its catalytic activity Measure the rate of individual catalytic events Rate the of catalytic reaction is proportionate to and a specific step in catalysis the amount of enzyme present in concentration The limited sensitivity of traditional enzyme assays necessities the use of the large group, or Enzyme Assays ensemble, of enzyme molecules in order to ELISA produce measurable precipitate Spectrophotometric Assay Coupling Dehydrogenase Competitive Ligand Binding Assay  If there is a protein that will bind the analyte, and Tissue damage result in an increased release of the bound and free analyte (ligand) can be intracellular enzymes into the plasma. The separated and measured. activities of many of these enzymes are routinely determined for diagnostic purposes in diseases Dry Chemistry Dipsticks of the heart, liver, skeletal muscle, and other For a number of assays, the enzymes or tissues. antibodies and reagents can be combined on a The level of specific enzyme activity in the plastic strip. plasma frequently correlates with the extent of For measurement of blood glucose, a finger- tissue damage. prick blood sample is placed on the test strip that Determining the degree of elevation of an contains glucose reagents. enzyme activity in the plasma is often useful in For urine testing, several different assays can be evaluating the prognosis of the patient. included as separate pellets on a plastic stick. Similar dipsticks are available to detect human Plasma Enzyme as Diagnostic Tools chorionic gonadotropin in urine. Some enzymes show relatively high activity in only one of a few tissues. Therefore, the Screening for Inborn Errors of Metabolism presence of increased levels of these enzymes in The concentration of offending amino acids is plasma reflects damage to the corresponding measured in a blood sample that is normally tissue. taken a week after birth, when the enzymes that ALT – Alanine aminotransferase is abundant in are affected in the disease. the liver. A significant increase in ALT indicates a The Guthrie bacterial inhibition test – the blood possible damage to the hepatic tissue. sample is laid onto an agar plate that has been seeded with a phenylalanine-requiring strain of GOOD TO KNOW: Measurement of ALT is part of the Bacillus subtilis, together with a competitive liver function test panel. inhibitor of phenylalanine uptake into the bacteria. The bacterial inhibition test has been superseded by chromatographic techniques that permit the detection of a variety of abnormal metabolites. Diagnostic value of Enzymes Plasma Enzymes Functional Plasma Enzyme Enzymes or proenzymes present in the plasma of a normal individual at all times and perform a physiologic function Produced and secreted by the liver Ex. lipoprotein lipase; proenzymes for blood coagulation Nonfunctional Plasma Enzyme Present in small amount and usually perform no known physiologic function Arise from routine normal destruction of RBC, WBC and other cells Increase in these enzyme tissue damage Plasma Enzyme Levels in Disease States Important Characteristics of Enzymes as a Diagnostic Lactate dehydrogenase (LDH) Tool At least five different isozymes Should be tissue specific Assess the timing and extent of heart damage Appear within the “Diagnostic Window” due to myocardial infarction (heart attack) 12 hrs of MI: blood level of total LDH increases, “Diagnostic window – a cardiac marker’s timeline for and there is more LDH2 than LDH1 rising, peaking, and returning to normal after a heart 24hrs of MI: more LDH1 than LDH2 attack” Amylase Activity increased in acute pancreatitis Normal levels: 0.2-1.5IU/I Peak value in 8-12hrs – onset of disease and returns to normal in 3-4 days Urine analysis Serum analysis – chronic pancreatitis, acute parotitis (mumps) and obstruction of Creatinine Phosphokinase (CPK) pancreatic duct Normal level: 10-50UI/I GOOD TO KNOW: Lipase is more specific than Amylase Diagnosis of MI – Very early detection, muscular for Acute Pancreatitis. dystrophy, hypothyroidism. Alcoholism Troponin Serum Glutamate Pyruvate Transferase (SGPT) Normal level: 0.04 ng/mL Also known as Alanine transaminase (ALT) Levels above 0.40 ng/mL indicates a probable Normal level: 3-4.0 IU/I heart damage Acute hepatitis of viral or toxic origin Troponin is a complex of three proteins present Jaundice and cirrhosis of liver in skeletal and cardiac muscles Increases for 2 to 6 hours after an MI and remain Serum Glutamate Oxaloacetate Transferase (SGOT) elevated for 4 to 10 days Also known as Aspartate transaminase Provides a sensitive and specific indication of Normal level: 4-4.5UI/I damage to heart muscle Increase in myocardial infarction and also in liver diseases Enzymes Facilitate the Diagnosis of Genetic Diseases SGPT is more specific for liver disease and SGOT Polymerase Chain Reaction (PCR) for MI – SGPT more cytosomal enzyme while Relies upon the ability of enzymes to serve as SGOT is cytosol and mitochondria catalytic amplifiers to analyze the DNA present in biologic and forensic samples Alkaline Phosphatase Produces thousands of copies of a defined Elevated in bone and liver disease segment of DNA from a minute quantity of Normal level: 25-90 IU/I starting material Diagnosis for rickets, hyperparathyroidism, Employs a thermostable DNA polymerase & carcinoma of bone, obstructive jaundice, Paget’s appropriate oligonucleotide primer to produce disease thousands of copies of a defined segment of DNA from a minute quantity starting material. Acid Phosphatase Use to detect and characterized DNA present Normal level: 0.5-4 KA units/dl initially at levels too low for direct detection. Increased in cancer of prostate gland and Paget’s Used for screening genetic mutation and Diseases detection of pathogens and parasites: Good tumor marker o Trypanosoma cruzi – Chagas disease GOOD TO KNOW: There are 5 isoenzymes for Acid o Nesseria meningitides – bacterial phosphates. meningitis Restriction Fragment Length Polymorphism (RFLP) Field in biochemistry concerned with Involve enzymes – Restriction Endonucleases quantitative measurement of the rates of o Cuts DNA in sites specified by a enzyme catalyzed reactions and systematic sequence of four or six or more base study of factors that affects the rate pairs (restriction site) o Deviation from the normal products can Chemical Reaction occur if there is mutation (Deviation → Substance → different substance RFLP) A+B→C+D o Occurs if a mutation renders a restriction - Breaking or making covalent bonds site unrecognizable to its cognate Reactants (substrate) restriction endonucleases or, Products → end results of the reaction alternatively, generates a new Reaction rate → speed with which the reaction recognition site. takes place - Change in concentration during a Facilitates prenatal detection of hereditary certain period of time disorders Concept of Free Energy Restriction endonuclease − Potential Energy stored in chemical bonds of a Enzymes that have cleave double – stranded molecule DNA at sites specified by a sequence of four, six or more base pair called restriction sites. In chemical reaction, 2 things can happen: Cleavage of a sample DNA with a restriction enzyme energy is transferred from one molecule to produces a characteristic set of smaller DNA fragments. another & use of energy from the reactant molecule to do work (chemical work, transport Tools for studying Enzymes work, mechanical work) Recombinant DNA Activation Energy Cloning the gene of the enzyme in E coli or yeast − Energy required to initiate a reaction → larger quantities of enzyme Free Energy Change Recombinant fusion proteins o Difference in the free energy of the o Create modified proteins (enzymes) reactants and products ▪ Achieved by linking the gene to an oligonucleotide sequence “His tag” – six consecutive histidine residues o can be purified by affinity chromatography Site Directed Mutagenesis Protein from the cloned gene is established → to identify the action or role of a specific aminoacyl residues→ Site Directed Mutagenesis (results to change in the codon of the specific aminoacyl residues) → provide insight into the mechanism Chemical Reaction of enzyme action Balanced - Substrate, products of a chemical reaction recorded on their correct proportion(stoichiometry) - A+B⇿P+Q - Reversible reaction ENZYME KINETICS Independent properties in a biochemical reaction Energy - G – represents the amount of energy released for required per mole of reactant - The amount or sign G indicates nothing about the rate of the reaction Rate - Determined by the energy of activation Relationship between Keq and Free Energy ▪ The energy required to initiate Free energy is negative = Keq > 1 the reaction - Favor Left to Right reaction - Conc. Of the product is higher than the substance at equilibrium Free energy change is positive = Keq < 1 - R to L - More substance at equilibrium is formed Free energy does not give information regarding the rate What determine the rate of chemical reaction? Change in Free Energy Describes 2 things - Direction of the chemical reaction - Concentration of the substrate and product at equilibrium Equal to the - Free energy of the formation of the products – free energy of the formation of the substrate -  G0 = Gp - Gs Activation Energy Free energy of formation is (+) in almost all G0 = free energy of the product < free energy chemical reaction of the substrate - Therefore, formation of transition state Direction: from left to right is an energy requiring chemical reaction Favors the formation of the product - The energy needed to form a transition Spontaneous reaction state is known as Activation Energy Therefore, the magnitude and the sign of the - The one that determine therefor the free energy determine how far the reaction will rate of the reaction will the value of the proceed  Gf Negative free energy – spontaneous reaction Positive Free energy – favors the right to left Factors that affect the rate of reaction reaction Kinetic Energy Theory (Collision Theory) - Formation of substrate from the - For 2 molecules to react they must products collide and possess the sufficient energy to overcome the energy barrier G0 = -RT In Keq Increase in temperature, increase energy - R (gas constant); T (absolute Temp)  Increase in concentration, increase the tendency Michaelis Menten Equation to collide Describes how the rate of the reaction (Vi) depends on the concentration of both enzymes Concentration of the reactants: [E] and the substrate [S] which forms the product [P] Involves – Rate of Enzyme catalyzed reaction (Vi) – Concentration of the substrate – 2 constants – Vmax 1st ORDER REACTION – Km The rate of the reaction is directly proportional to the amount of the substrate, therefore a illustrate the relationship between the between linear relationship the initial velocity of the reaction and substrate ZERO-ORDER Reaction concentration The velocity of the reaction is constant and independent of substrate concentration Enzymes do not affect the Keq Enzymes accelerate the reaction rate by lowering the activation barrier Gf Initial rate of the reaction – Trace of the product is present o no feedback inhibition o negligible reverse reaction going on – rate of the forward reaction – is proportionate to the concentration of the enzyme Factors that affect the rate of Enzyme Catalyzed reaction Temperature – Increases the rate of the reaction but can also increase the kinetic energy of the enzyme >> denaturation of enzyme >> slow down the rate Hydrogen Ion Concentration – Enzymes in the body exhibit optimum activity at pH values between 5-9 At the Initial Rate of the Equation, the trace of the product is present. There is no feedback inhibition and there is negligible reverse reaction going on. The rate of the forward reaction is proportionate to the concentration of the enzyme. Michaelis Constant (Km) Is the substrate concentration at which Vi is half the maximal velocity (Vmax/2) attainable at a particular concentration of enzyme Double Reciprocal or Lineweaver-Burk Plot At point A, the conc of the substrate [S] > Km, enzyme → equal to the catalytic constant making the initial velocity (Vi) equal to Vmax. (Rate Turnover number - the no. of moles of is unaffected by further increase in substrate since substrate that reacts to form product the enzymes are fully saturated) per mole of enzyme per unit time Reflects the efficiency of At point B, the conc of the substrate is equal to ½ enzymatic catalysis Vmax equal to the catalytic constant Significance of Vmax Significance of Michaelis Constant At Vmax the enzyme is fully saturated with the The affinity of an enzyme for its substrate is the substrate inverse of the dissociation constant Kd for the The reaction is proceeding at its maximum rate dissociation of the enzyme substrate complex (Vmax can be improved if you add more ES enzymes) Km approximates Kd – The higher the Km the less tightly the Linear Form of Michaelis-Menten Equation substrate is bound to the enzyme Permits Vmax and Km to be extrapolated from the initial velocity data obtained at less than saturating concentration of the subject Cooperative Binding Encountered in allosteric or multimeric Double Reciprocal or Lineweaver-Burk Plot enzymes Vi = Vmax [S]/Km+[S] – Enzymes that are capable of binding Invert with substrate at multiple sites 1/Vi= Km+[S]/Vmax[S] The relation between the changes in the initial Factor velocity and the changes in the concentration of 1/Vi = Km/Vmax [S] + S/Vmax [S] the substrate is sigmoidal 1/Vi = (Km/Vmax) (1/S) + 1/Vmax – Hill Equation Significance of the Hill Coefficient ENZYME INHIBITORS Empirical parameter whose value is the function − Substances that interfere with the action of an of number, kind, and strength of the enzyme and slows down the rate of a reaction interactions of the multiple substrate binding Classification of the action of Inhibitors sites on the enzymes – Chemical Modification n=1 Reversible/Irreversible – All the binding site behave Inhibition independently – Kinetic Influence n>1 Competitive/ Noncompetitive – Exhibit cooperativity inhibition Binding with of the first Raising the substrate may or substrate molecule will may not overcome inhibition enhance the affinity of the enzyme for binding to Influence on the Chemical Modification of enzymes additional substrate Reversible Inhibitors The greater the value of n the greater the Binds to the enzyme and subsequently be degree of cooperativity released, leaving the enzyme in its original condition Irreversible Inhibitor Reacts with the enzyme to produce an protein that is not enzymatically active and from which the original enzyme cannot be regenerated Involves making or breaking covalent bonds with aminoacyl residues essential for substrate binding, catalysis, or functional conformation of the enzyme IMPORTANT CONCLUSIONS Influence on the Kinetic Activity of the Enzyme Characteristics of Km 1. Reflects the affinity of the enzyme for that substrate 2. Is numerically equal to the substrate concentration at which the reaction velocity is equal to ½ Vmax 3. Small Km – high affinity to the enzyme to the substrate 4. Large Km – low affinity of the enzyme to the substrate Multimeric enzymes - Enzymes that bind substrates at multiple sites - Substrate binding exhibits cooperativity ▪ S50 - the substrate concentration that results in Competitive Inhibition half-maximal velocity Inhibitors have very similar structure with the substrate analogs Binds with the active site and blocks the substrate’s access to it Effects can be overcome by increasing the [S]  Has no effect on the Vmax but raises the Km for the substrate Inhibitor binds reversibly with enzyme in the active site Effect on Vmax o At sufficiently high substrate concentration the reaction velocity reaches the Vmax Effect on Km o Increases the apparent Km of the substrate. In the presence of competitive inhibitor more substrate is needed to reach the Vmax Effect on the Lineweaver-Burk Plot Sequential Reaction o Inhibited and uninhibited reactions Involves 2 or more substrate show different x axis intercepts but Both substrates must be combined with the same y axis intercepts enzyme to form a ternary complex before catalysis can proceed Noncompetitive Inhibitor Random Sequential reaction Inhibitor binds with enzyme at a site other than – Either substrates may combine first the active site; change in the structure of the with the enzyme enzyme esp. around the active site modification Compulsory of catalytic activity – Specific order of combining with Does not affect the binding of the substrate enzyme Bear little or no resemblance to substrate Lowers the Vmax but does not affect Km Ping-Pong Reaction One or more products are released from the Acts on the outside of the active site (example enzyme before all the substrates are added acts with the substrate or acts on the enzyme to destroy it to remove the competition) Regulation of Enzyme Activity 1. Passive Effect on Vmax − Regulation of the Substrate Concentration o Decreases the Vmax o Response to the changes in substrate Effect on Km level o No change in the Km o Intracellular level of most substrates is Effect on the Lineweaver-Burk Plot in the range of their Km o Inhibited and uninhibited reactions o Limited scope show same x axis intercepts but 2. Active different y axis intercepts A. Compartmentalization of Enzyme a. Specific metabolic pathway takes place in specific cellular compartments b. Controlling an Enzyme that Catalyzes a rate-limiting reaction B. Regulation of Enzyme Quantity a. Control of synthesis b. Control of Enzyme Degradation C. Regulation of Enzyme Catalytic activity a. Allosteric regulation b. Covalent modification 3. Controlling the enzyme that catalyzes the rate − Effects of the “effectors” limiting step a. Effectors Product of one reaction will serve as substrate ▪ affect the affinity of the enzyme for the next reaction → each coupled reaction to the substrate has a free energy change which is negative ▪ Modify the catalytic activity of Enzyme that catalyzes a step in the metabolic the enzyme pathway that is relatively slower compared to b. The effectors the rest i. Negative - inhibit enzyme Results to decrease production effector Mechanism which is exploited by ii. Positive - increases enzyme pharmaceutical companies activity a. HMG-CoA reductase → synthesis of Allosteric enzyme usually contains multiple subunits cholesterol and frequently catalyze the committed step in the pathway Regulation of Enzyme Quantity The absolute quantity of an enzyme reflects the Feedback inhibition net balance between the rate of synthesis and − Kinetics – may be competitive, noncompetitive, its rate of degradation partially competitive, or mixed Enzymes subject to regulation of synthesis are − Typically inhibit the first committed step often those that are needed at only one stage of development or under selected physiologic Allosteric enzymes condition − Enzymes whose activity may be modulated by the presence of effectors in the allosteric site Regulation of the amount of enzyme can be achieve by: K-series Allosteric enzymes Control rate of enzyme synthesis ▪ Substrate saturation kinetics is Increased synthesis (Induction) competitive Decreased synthesis (repression) ▪ Km is increased ▪ Note: Concentration of enzyme ▪ Conformational change in the may be: active site results in the ▪ Constant weakening of bonds between ▪ Constitutive substrate and substrate binding Control of Degradation residue Ubiquitous proteasome pathway V-series Allosteric enzyme Control of Enzyme synthesis can be induced or ▪ Allosteric inhibitors decrease reduced (involves the DNA sequence) the Vmax ▪ Conformational change in the Inducer ▪ Substrate or structurally related active site is due to alteration in compounds the orientation or charge of the Repressor catalytic residues ▪ Excess of metabolite 2 types of effectors Homotrophic Effectors Substrate itself serves as an effector Regulation of the Catalytic Activity The presence of the substrate molecule at one site on the enzyme enhances the Allosteric regulation catalytic properties of the other a. Regulation is achieved by binding with substrate binding site 􀀀 exhibit dissociable cooperativity When its activity is plotted - V0 vs [S] Allosteric binding site follows a sigmoidal curve − Allosteric enzyme is regulated by molecules Heteroptrophic Effectors called effectors that binds noncovalently at a site other than the active site Effector is different from the substrate Regulatory Covalent Modification Irreversible – Partial proteolysis Reversible – Phosphorylation Allosteric enzyme usually contains multiple subunits and frequently catalyze the committed a step in the pathway Regulation by Covalent modification Phosphorylation and Dephosphorylation o Catalyzed by protein kinase Altering the activity of an enzyme via the covalent bonding of another molecule to the enzyme; most often accomplished by the addition/subtraction of phosphate groups’ thus the enzyme could more or less active in the phosphorylated form Regulatory Covalent Modification Irreversible – Partial proteolysis Reversible – Phosphorylation