Unit-III Enzymes
Contents 1. Introduction and Properties of enzymes 2. Nomenclature and Classification 3. Mechanism of enzyme-catalyzed reactions 4. Kinetics of enzyme-catalyzed reactions 5. Inhibition of enzymes 6. Regulation of enzymes 7. Clinical applications of enzymes
Introduction and Properties of Enzymes
Enzymes Enzymes are catalysts that have special characteristics to facilitate the biochemical reactions in the biological systems. Enzyme-catalyzed reactions take place usually under relatively mild conditions.
Catalyzed reactions Reactants need to pass over the energy barrier, ΔG +. Catalysts reduce the activation energy and assist the reactants to pass over the activation energy.
Need for special catalysts Chemical reactions in living systems are quite different from that in the industrial situations because of 1. Fragile structures of the living systems 2. Low kinetic energy of the reactants 3. Low concentration of the reactants 4. Toxicity of catalysts 5. Complexity of the biological systems
Characteristics Enzyme-catalyzed reactions have the following characteristics in comparison with the general catalyzed reactions: common features: 2 do and 2 don t Do not consume themselves: no changes in quantity and quality before and after the reactions. Do not change the equilibrium points: only enhance the reaction rates. Apply to the thermodynamically allowable reactions Reduce the activation energy unique features: 3 high Enzyme-catalyzed reactions have very high catalytic efficiency. Enzymes have a high degree of specificity for their substrates. Enzymatic activities are highly regulated in response to the external changes.
Classification Oxidoreductases Transferases Hydrolases Lysases Isomerases Ligases
Conventional Nomenclature Adding the suffix ase to the name of the substrates (urease) Adding the suffix ase to a descriptive term for the reactions they catalyze (glutemate dehydrogenase) For historic names (trypsin, amylase) Being named after their genes (Rec A reca, HSP70)
Systematic Nomenclature The International Union of Biochemistry and Molecular Biology (IUBMB) maintains the classification scheme. Categorize in to 6 classes according to the general class of organic reactions catalyzed Assigned a unique number, a systematic name, a shorter common name to each enzyme
High specificity Unlike conventional catalysts, enzymes demonstrate the ability to distinguish different substrates. There are three types of substrate specificities. Absolute specificity Relative specificity Stereospecificity
Absolute specificity Enzymes can recognize only one type of substrate and implement their catalytic functions. NH 2 urease O C NH 2 + H 2 O 2NH 3 + CO 2 urea NH CH 3 O C + H 2 O NH 2 methyl urea
Relative specificity Enzymes catalyze one class of substrates or one kind of chemical bond in the same type. protein kinase A protein kinase C protein kinase G To phopharylate the -OH group of serine and threonine in the substrate proteins, leading to the activation of proteins.
Stereospecificity The enzyme can act on only one form of isomers of the substrates. H H H 3 C C OH COOH H 3 C C COOH OH A B C A B C Lactate dehydrogenase can recognize only the L-form but the D-form lactate.
High regulation Enzyme-catalyzed reactions can be regulated in response to the external stimuli, satisfying the needs of biological processes. Regulations can be accomplished through varying the enzyme quantity, adjusting the enzymatic activity, or changing the substrate concentration.
Components of Enzymes
Active Center Almost all the enzymes are proteins having well defined structures. Some functional groups are close enough in space to form a portion called the active center. Active centers look like a cleft or a crevice. Active centers are hydrophobic.
Lysozyme Residues (colored ) in the active site come from different parts of the polypeptide chain.
Two essential groups The active center has two essential groups in general. Binding group: to associate with the reactants to form an enzyme-substrate complex Catalytic group: to catalyze the reactions and convert substrates into products
Protein chain Essential groups outside the active center Substrate molecule + - Catalytic group Active center Binding group
Active centers
Molecular Components Simple enzymes: consists of only one peptide chain Conjugated enzymes: holoenzyme = apoenzyme + cofactor (protein) (non-protein) Cofactors: metal ions; small organic molecules
Metal ions Metal-activated enzyme: ions necessary but loosely bound. Often found in metalactivated enzyme. Metalloenzymes: Ions tightly bound. Particularly in the active center, transfer electrons, bridge the enzyme and substrates, stabilize enzyme conformation, neutralize the anions.
Organic compounds Small size and chemically stable compounds Transferring electrons, protons and other groups Vitamin-like or vitamin-containing molecule
Coenzymes Loosely bind to apoenzyme. Be able to be separated with dialysis. Accepting H + or group and leaving to transfer it to others, or vise versa. Prosthetic groups Tightly bind through either covalent or many non-covalent interactions. Remained bound to the apoenzyme during the course of reaction.
Mechanism of Enzyme- Catalyzed Reactions
Lock-and-key model Both E and S are rigid and fixed, so they must be complementary to each other perfectly in order to have a right match.
Induced-fit model The binding induces conformational changes of both E and S, forcing them to get a perfect match.
Induced structural changes
Kinetics of Enzyme- Catalyzed Reactions
Intermediate state Forming an enzyme-substrate complex, a transition state, is a key step in the catalytic reaction. E + S k 1 k 3 k 2 ES E+P initial intermediate final
Rate constants E + S k 1 k 3 k 2 ES E+P K 1 = rate constant for ES formation K 2 = rate constant for ES dissociation K 3 = rate constant for the product released from the active site
Michaelis-Menten Equation The mathematical expression of the product formation with respect to the experimental parameters Michaelis-Menten equation describes the relationship between the reaction rate and substrate concentration [S].
Assumptions [S] >> [E], changes of [S] is negligible. K 2 is negligible compared with K 1. Steady-state: the rate of E-S complex formation is equal to the rate of its disassociation (backward E + S and forward to E + P)
[S] V = K m + [S] V max Describing a hyperbolic curve. K m is a characteristic constant of E [S] << K 时,v [S] m [S] >> K 时,v m V max
V 0 V max Zero order with respect to [S] First order with respect to [S] 0 [S]
Significance of K m the substrate concentration at which enzyme-catalyzed reaction proceeds at one-half of its maximum velocity K m is independent of [E]. It is determined by the structure of E, the substrate and environmental conditions (ph, T, ionic strength, )
V 0 V max V max /2 0 [S] K m
K m is a characteristic constant of E. The value of K m quantifies the affinity of the enzyme and the substrate under the condition of K 3 << K 2. The larger the K m, the smaller the affinity. k 2 + k 3 K m = k 1
Significance of V max The reaction velocity of an enzymatic reaction when the binding sites of E are saturated with substrates. It is proportional to [E].
Factors affecting enzyme-catalyzed reaction Substrate concentration Enzyme concentration Temperature ph Inhibitors Activators
Inhibition of Enzyme
Inhibitors Inhibitors are certain molecules that can decrease the catalytic rate of an enzyme-catalyzed reaction. Inhibitors can be normal body metabolites and foreign substances (drugs and toxins).
Inhibition processes The inhibition process can be either irreversible or reversible. The inhibition can be competitive, noncompetitive, or un-competitive.
Summary of inhibition type binding target K m V max Competitive E only = Noncompetitive E or ES = Uncompetitive ES only
Activators Metal ions essential activators: no enzymatic activity without it Mg 2+ of hexokinase non-essential activators: enhancing the catalytic power.
Cofactors Cofactors Essential ions Coenzymes Activator ions (loosely bound) Metal ions of metalloenzymes (tightly bound) Cosubstrates (loosely bound) Prosthetic groups (tightly bound)
Essential ions Activator ions: loosely and reversibly bound, often participate in the binding of substrates. Metal ions of metalloenzymes: tightly bound, and frequently participate directly in catalytic reactions.
Function of metal ions Transfer electron Linkage of S and E; Keep conformation of E-S complex Neutralize anion
Coenzymes Act as group-transfer reagents to supply active sites with reactive groups not present on the side chains of amino acids Cosubstrates: Prosthetic groups:
Cosubstrates The substrates in nature. Their structures are altered for subsequent reactions. Shuttle mobile metabolic groups among different enzyme-catalyzed reactions.
Prosthetic groups Supply the active sites with reactive groups not present on the side chains of AA residues. Can be either covalently attached to its apoenzymes or through many noncovalent interactions. Remained bound to the enzyme during the course of the reaction.
Regulation of Enzyme
Many biological processes take place at a specific time; at a specific location and at a specific speed. The catalytic capacity is the product of the enzyme concentration and their intrinsic catalytic efficiency. The key step of this process is to regulate either the enzymatic activity or the enzyme quantity.
Reasons for regulation Maintenance of an ordered state in a timely fashion and without wasting resources Conservation of energy to consume just enough nutrients Rapid adjustment in response to environmental changes
Controlling an enzyme that catalyzes the rate-limiting reaction will regulate the entire metabolic pathway, making the biosystem control more efficient. Rate limiting reaction is the reaction whose rate set by an enzyme will dictate the whole pathway, namely, the slowest one or the bottleneck step.
Regulation of Enzyme Activity Zymogen activation Allosteric regulation Covalent modification
Clinical Applications
Fundamental Concepts Plasma specific or plasma functional enzymes: Normally present in the plasma and have specific functions. High activities in plasma than in the tissues. Synthesized in liver and enter the circulation. Impairment in liver function or genetic disorder leads to a fall in the activities.
Non plasma specific or plasma nonfunctional enzymes: either totally absent or at a low concentration in plasma In the normal turnover of cells, intracellular enzymes are released into blood stream. An organ damaged by diseases may elevate those enzymes
Isoenzyme A group of enzymes that catalyze the same reaction but differ from each other in their structure, substrate affinity, V max, and regulatory properties. Due to gene differentiation: the different gene products or different peptides of the same gene Present in different tissues of the same system, or subcellular components of the same cell (e.g) Lactate dehydrogenase (LDH) Creatine phosphokinase
Diagnostic Applications Usefulness: Enzyme assays provide important information concerning the presence and severity of diseases Provide a means of monitoring the patient s response approaches: Measuring the enzymatic activities directly Used as agents to monitor the presence of substrates
Enzymes for disease diagnosis Amylase Serum enzymes (elevated) Serum glutamate pyruvate transaminase (SGPT) Serum glutamate oxaloacetate transaminase (SGOT) Alkaline phosphatase Acid phosphatase Lactate dehydrogenase (LDH) γ-glutamyl transpeptidase (GGT) 5 -nucleotidase Aldolase Diseases Acute pancreatitis Liver diseases (hepatitis) Heart attack (myocardial infarction) Rickets, obstructive jaundice Cancer of prostate gland Heart attack, liver diseases Alcoholism Hepatitis Muscular dystrophy
Therapeutic Applications Successful therapeutic uses Steptokinase: treating myocardial infarction; preventing the heart damage once administrated immediately after heart attack Asparaginase: tumor regression Several limits Can be rapidly inactivated or digested May provoke allergic effects