Enzymes PDF

**Enzymes** **Outline of Lectures** - What enzymes are, and why they do - Enzyme structure & classification, and enzyme co-factors - Enzymes & cellular metabolism - How enzymes work, and the factors that affect enzyme function - How enzymes interact with their substrates - Enzyme...

**Enzymes** **Outline of Lectures** - What enzymes are, and why they do - Enzyme structure & classification, and enzyme co-factors - Enzymes & cellular metabolism - How enzymes work, and the factors that affect enzyme function - How enzymes interact with their substrates - Enzyme kinetics - Enzyme inhibition **Learning Outcomes** - Describe the structure, classification & function of enzymes - Describe the metabolic processes that are catalysed by enzymes, and why enzyme catalysis is needed - Describe the nature of the interaction between enzymes & their substrates - Describe enzyme kinetics & the associated Michaelis-Menten & Lineweaver-Burk Plots - Describe the various types of enzyme inhibition - Appreciate the relevance of enzymes & enzyme inhibition in medicine **Enzymes** - **What are enzymes?** - Specialised, catalytically active biological macromolecules - Act as specific, efficient and active catalysts of chemical reactions in aqueous solution - Most enzymes are a. **Globular proteins** b. **Some are RNA -- e.g. ribozymes and ribosomal RNA** nucleotides - **How are enzymes named and classified?** - Enzymes are named by adding the suffix **"-ase"** to **the name of their substrate *[or]* a word or phrase describing their catalytic action** - Classification based on the type of reaction catalysed **Classification** **Type of Reaction Catalysed** -------------------- -------------------------------------------- Oxidoreductases Oxidation -- reduction reactions Transferases Transfer of functional groups Hydrolases Hydrolysis reactions Lyases Group elimination to form double bonds Isomerases Isomerization Ligases Bond formation coupled with ATP hydrolysis A table with different types of cells Description automatically generated - **Naming of Enzymes** - Each enzyme is assigned a 'four-part classification number' and 'a systematic name', which identifies the reaction it catalyses -- *e.g. for hexokinase:* a. Formal name: ATP:D-hexose 6-phosphotransferase transfer phosphoryl groups into glucose? b. Enzyme commission number is 2.7.1.1 - 2 = the class name (transferase) - 7 = subclass (phosphotransferase) - 1 = phosphotransferase with a hydroxyl group as acceptor - 1 = D-glucose as the phosphoryl group acceptor - **What are the key structure-function features of enzymes?** - Enzymes are protein - They have a globular shape & a complex 3-D structure within this structure -- active site - They have an **'active site'** -- its unique shape & chemical environment determine which substrate(s) will bind → determine the specificity of enzyme - Some enzymes require additional non-protein chemical component(s), called **cofactors(s)**, in order to function properly protein component itself is not active, need cofactors -- activate enzyme! - Cofactors act as non-protein "helper" molecules -- may be **metal ions** or **organic** / **metallo-organic molecules**  most of the metallic ions' coenzyme are vitamins! - **Metal ion cofactors** - Small inorganic ions - [Mg^2+^, *K*^+^, Ca^2+^, Zn^2+^, Cu^2+^, *Co*, *Fe* ]{.math.inline} - May be free (e.g. [Na^+^, *K*^+^]{.math.inline}) or held in coordination complexes with the enzyme protein (e.g. [Zn^2+^, Ca^2+^]{.math.inline}) actually bound to the enzyme / metal ions incorporates with it -- show catalytic actions - Assist with enzyme catalysis A table with many types of substances Description automatically generated with medium confidence - **Organic / metallo-organic cofactors** - Coenzymes -- organic cofactors that are **loosely** bound and easily released from the enzymes - Prosthetic groups -- organic cofactors that are **tightly** bound to the enzymes - Coenzymes usually act as 'co-substrates' or as transient carriers of specific functional groups - Most are derived from **vitamins** -- organic nutrients that are required in small amount in the diet - Examples include: a. **NAD (niacin; B3)** b. **FAD (riboflavin; B2)** c. **Coenzyme A** **Enzymes and Cofactors** - The complete, catalytically active enzyme together with its bound coenzyme and/or metal ion is called ***holoenzyme*** → active enzyme - The protein part of such enzyme is called the ***apoenzyme*** or ***apoprotein*** (inactive)  **Enzymes** - **Why enzymes so important?** - They catalyse (accelerate) biochemical reactions slow reactions in the body -- by speeding up chemical reactions - Most biochemical & physiological reactions in the body proceed at very slow pace - Enzymes act to speed up these so-called 'chemical reactions of life' - Without enzymes, most chemical reactions of life would proceed so slowly (or not at all) that life could not exist - **Rules of Catalysis** 1. A catalyst cannot catalyse a thermodynamically unfavourable reaction 2. A catalyst cannot change the course of a reaction 3. A catalyst cannot change the equilibrium of a reaction, only the rate at which equilibrium is reached. It lowers the activation energy for the reaction 4. A catalyst may exert a directing influence. If two reactions are thermodynamically possible (A going to B or C) and a catalyst only catalyses one of them, then that reaction will be favoured 5. A catalyst is recoverable, so only small amounts are necessary - **What are these 'Chemical Reactions of Life' that are catalysed by enzymes?** - Enzymes catalyse cellular metabolic reactions - Metabolism is the sum of the chemical reactions that take place in an organism - Two types of metabolism or metabolic reactions a. **Anabolism or Anabolic reactions -- involve the formation of bonds between molecules** ⇒ Catalysed by Anabolic enzymes - Biosynthetic -- **building** of **complex** molecules from simpler ones - Involve the formation of bonds between molecules → requires energy from ATP to proceed - Energy-utilising processes/reactions - Involve ***dehydration synthesis*** reactions (reactions that release water) -- **e.g. carbohydrate/protein synthesis** ㄴ**Endergonic** -- consume more energy than they produce b. **Catabolism or Catabolic reactions -- involve the breaking of bonds between molecules** ⇒ Catalysed by Catabolic enzymes - Degradative -- **breakdown** of complex molecules into simpler ones - Involve the breaking of bonds between molecules → release energy -- restored in ATP - Energy-releasing processes/reactions - Involve ***hydrolytic*** reactions (use water to break chemical bonds) -- **e.g. digestion of carbohydrates** ㄴ**Exergonic** -- produce more energy than they consume **Cellular metabolism** A diagram of a complex reaction Description automatically generated **Enzyme-catalysed Metabolic Reactions** - **Anabolic -- dehydration synthesis (synthesis)**  → release of water - **Catabolic -- hydrolysis (digestion)** A diagram of a chemical formula Description automatically generated **Enzymes and Cellular Metabolism** - **So, why do cellular metabolic reactions require the intervention of enzymes?** - All chemical/metabolic reactions require the initial input of energy **(Activation Energy,** [**E**~**A**~]{.math.inline}**)** in order to proceed (slow reaction - they usually cannot get energy that is required to proceed) - [*E*~*A*~]{.math.inline} is needed a. to increase collisions between reactant molecules b. to shift the reactant molecules into a **'transition state'**, where existing bonds can be broken & new ones formed - [*E*~*A*~]{.math.inline} is usually too high for the metabolic reactions to proceed significantly at ambient temperature - Enzymes, as catalysts, help to **lower the** [**E**~**A**~]{.math.inline} and enable metabolic reactions to proceed at a faster rate  → need energy to initially start the reaction! Endergonic -- loads of input energy to start reaction / release less energy after they form product Exergonic -- small activation energy required / large energy is produced for the products **Enzyme-catalysed Metabolic Reactions** **How do enzymes work?** - They act as catalysts -- speed up metabolic/biochemical reactions without being consumed or chemically altered reuse for another catalytic reactions - They provide an alternative pathway or mechanism for the reaction & lower the activation energy, [*E*~*A*~]{.math.inline} - They bind to & form an intermediate with the reactant (substrate), which is released later on during the product formation step helps substrate to get to the "transition state" - Enzymes accelerate the rate of the reaction without shifting or changing the equilibrium of the reaction! ⇒ Equilibrium is reached faster with enzyme! A diagram of a complex Description automatically generated with medium confidence  reduction in activation E with enzyme A diagram of a function Description automatically generated  ㄴcatalysed rate is increasing massively with enzyme according to the table - **Enzymes bind their substrates with high specificity** - Binding specificity is governed by 3D arrangement of atoms on the active binding site A diagram of a cell structure Description automatically generated - **'Lock and Key' Model** - Simplistic model of enzyme action - Substrate fits into 3D structure of enzyme active site a. Weak chemical bonds formed between substrate and enzyme b. Like a "key fits into lock"  A diagram of a structure Description automatically generated - **'Induced Fit' Model** not necessarily need to completely fit -- active site undergoes shift to fit with the substrate - More accurate model of enzyme action - Substrate binding causes the enzyme to change shape ('conformational change), leading to a tighter fit a. This brings chemical groups in position to catalyse reaction  enzyme changes once it's bind with substrateA close-up of a molecule Description automatically generated **Enzymes** - **What are the factors that affect enzyme function?** - **Enzyme concentration** a. Initially, as ↑ enzyme concentration ⇒ ↑ reaction rate - More enzymes more molecules to interact ⇒ more frequent collisions with substrate b. Then, reaction rate levels off with further increase in enzyme concentration - Substrate concentration becomes the limiting factor - Not all enzyme molecules can find a substrate  - **Substrate concentration** a. Initially, as ↑ substrate concentration ⇒ ↑ reaction rate - More substrate ⇒ more frequent collisions with enzyme b. Then, reaction rate levels off with further increase in substrate concentration - All enzyme active sites become engaged **(saturated)** - Maximum rate of reaction has been reached (to the saturation point) A screen shot of a diagram Description automatically generated - **Temperature** a. ↑ temperature ⇒ ↑ reaction rate - Molecules move faster ⇒ ↑ collisions between enzyme & substrate b. ↓ temperature ⇒ ↓ reaction rate - Molecules move slower ⇒ ↓ collisions between enzyme & substrate c. **Optimum Τ°** -- peak effect on enzyme-catalysed reaction - Greatest number of molecular collisions of enzyme & substrate d. ↑ temperature beyond Optimum Τ° ⇒ **enzyme denaturation** & no longer function in catalysis - Disrupts bonds in enzyme & between enzyme and substrate - Enzymes lose their 3D shape (3° structure) → optimum temperature varies with enzymesA diagram of a normal curve Description automatically generated most of human enzymes -- show maximum reaction rate in body temperature - **pH** a. Changes in pH - Add or remove [*H*^+^]{.math.inline} ⇒ small changes in the charges on the enzyme & substrate molecules **(altered critical ionization states)** - Affect the binding of the substrate with the enzyme active site b. **Optimum pH** -- peak effect on enzyme-catalysed reaction - pH 6-8 for most human enzymes -- depends on localised conditions - e.g. pepsin (stomach) = **pH 2-3**; trypsin release from pancreas / digestion (small intestines) = **pH 8** c. Extreme pH levels ⇒ **enzyme denaturation** - Disrupt attraction between charged amino acids - Disrupt bonds & the enzyme's 3D shape - Active site is distorted ⇒ loss of substrate fit  human body -- slightly alkaline - **Salinity** a. Changes in salinity - Add or remove cations & anions can't interact without radical groups ? b. Extreme salinity ⇒ **enzyme denaturation** - Enzymes are intolerant of extreme salinity - Disrupts attraction between charged amino acids - Disrupts bonds & the enzyme's 3D shape - Affects 2° & 3° enzyme structure **Enzyme Kinetics** - Enzyme kinetics is the study of the rates of chemical reactions that are catalysed by enzymes - It provides insight into - the mechanisms of enzyme catalysis & their role in metabolism - how the activity of enzymes is controlled in the cell need to be tightly controlled - how drugs and poisons can inhibit or modulate the activity of enzymes (Aspirin -- COX enzyme ?) - In 1913, Michaelis and Menton proposed the model known as Michaelis-Menten Kinetics to account for & explain - how enzymes can increase the rate of metabolic reactions - how the reaction rates depend on the concentration of enzyme & substrate**\ ** **Michaelis-Menten Kinetics** - All enzymes show a 'saturation effect; with their substrates - At low substrate concentration \[S\], the reaction rate or velocity, V Is proportional to \[S\] - However, as \[S\] is increased, the reaction rate falls off, and is no longer proportional to \[S\] - On further increase in \[S\], the reaction rate or velocity becomes constant and independent of \[S\] - At this stage, the enzyme is saturated with substrate - A plot of initial reaction velocity, V against substrate concentration \[S\] gives a rectangular hyperbola - The Michaelis-Menten equation or kinetics model was developed to explain or account for this 'saturation effect' - Relationship between Reaction Velocity and Substrate Concentration  - Michaelis and Menten proposed the following mechanism for a saturating enzyme-catalysed single substrate reaction: A close-up of a number of words Description automatically generated  - According to this postulate/scheme, - In an enzyme-catalysed reaction, a free enzyme, E binds its substrate, S to form an enzyme-substrate complex, ES - ES either undergoes further transformation to yield a final product, P and the free enzyme, E or breakdowns via a reverse reaction to form the free enzyme, E and substrate, S - By re-arranging the equations, and making several assumptions, they derived the Michaelis-Menten Equation A mathematical equation with black text Description automatically generated Where, a. [*V*~max~]{.math.inline} = the maximum velocity or rate of reaction, at maximum (saturating) concentrations of the substrate b. [*K*~*M*~ = (*k*~ − 1~+*k*~2~)/*k*~1~]{.math.inline} = substrate concentration at which the reaction velocity is 50% of the [*V*~max~]{.math.inline} (Michaelis constant) (backward + product)/original? c. \[S\] = concentration of the substrate, S - A graph of initial reaction velocity, [*V*~0~]{.math.inline} against substrate concentration, \[S\] results in a rectangular curve, where [*V*~max~]{.math.inline} represents the maximum reaction velocity  **Linear Transformations of Michaelis-Menten Equation** - It is not easy to accurately determine [*V*~max~]{.math.inline} at high substrate concentrations from the Michaelis-Menten curve - Algebraic transformation of the Michaelis-Menten equation into linear forms for plotting experimental data - **Lineweaver-Burk Plot** (also called **Double Reciprocal plot**) a. Derived by taking the reciprocal of both sides of the Michaelis-Menten equation and separating out the components of the numerator on the right side of the equation: A mathematical equation with a plus and minus Description automatically generated  → This is equivalent to the equation of a straight line: **y = mx + c** - **Eadie-Hofstee Plot** a. Derived by inverting the Michaelis-Menten equation, and multiplying both sides of the equation by [*V*~max~]{.math.inline} b. A plot of V against V/\[S\] yields [*V*~max~]{.math.inline} as the y-intercept, [*V*~max~/*K*~*M*~]{.math.inline} as the x-intercept, and [*K*~*M*~]{.math.inline} as the negative slope **y = mx + c** A close-up of a mathematical equation Description automatically generated  **Significance of** [**K**~**M**~]{.math.inline} **(Michaelis Constant)** - It has same unit as the substrate concentration (μM) - The substrate concentration \[S\] at which the reaction proceeds at half maximal velocity (50%), i.e. [*K*~*M*~=]{.math.inline} \[S\] at ½ [*V*~max~]{.math.inline} - A measure of an enzyme's affinity for its substrate -- **the lower the** [**K**~**M**~]{.math.inline} **value, the higher the enzyme's affinity** for the substrate and vice versa - Provides an idea of the strength of binding of the substrate to the enzyme molecule -- **the lower the** [**K**~**M**~]{.math.inline} **value, the more tightly bound the substrate** is to the enzyme for the reaction to be catalysed - Indicates the lowest concentration of the substrate \[S\] the enzyme can recognise before reaction catalysis can occur - Describes the substrate concentration at which half the enzyme's active sites are occupied by substrate A screenshot of a chart Description automatically generated **chymotrypsin** **Significance of** [**V**~**max** ~]{.math.inline} - Gives an idea of **how fast the reaction can occur under ideal circumstances** - Reveals the turnover number of an enzyme, i.e. the number of substrate molecules being catalysed per second - Magnitude varies considerably -- from \ - **The apparent** [**K**~**M**~]{.math.inline} **is increased** - **There is no change in the** [**V**~**max** ~]{.math.inline} - The inhibition can be reversed by increasing the concentration of the substrate (S) A diagram of a cell cycle Description automatically generated  ㄴincreasing the concentration of substrate -- but same maximum reaction rate A diagram of a function Description automatically generated **Non-competitive Inhibition** - The inhibitor has similar affinity for both the free enzyme (E) and the enzyme-substrate complex (ES) - The inhibitor binds with the enzyme at a site which is distinct from the substrate binding site - The binding of the inhibitor **[does not]** affect the substrate binding, and vice versa - However, the inhibitor binding to enzyme or enzyme-substrate complex prevents enzyme from forming its product once its binding -- stabilise / not change - **There is no effect on, or change, in the** [**K**~**M**~]{.math.inline} - **The** [**V**~**max** ~]{.math.inline} **for the reaction is decreased**  A graph of a number of substances Description automatically generated with medium confidence Can bind either E or ES -- prevent the conversion to product / max reaction is reduced in the presence of I  **Uncompetitive Inhibition** - The inhibitor has affinity for the enzyme-substrate complex (ES), but not the free enzyme (E) - The inhibitor binds only (and stabilise) the enzyme-substrate complex (ES), but not the free enzyme (E) - An inactive ESI complex is formed when the inhibitor reversely binds to the enzyme-substrate complex - The inactive ESI complex does not form a product (P) - **The apparent** [**K**~**M**~]{.math.inline} **is decreased -- due to the selective binding of the inhibitor to the enzyme-substrate complex (ES)** - **The** [**V**~**max** ~]{.math.inline} **for the reaction is also decreased** - Inhibition cannot be reversed by increasing the substrate concentration A diagram of a chemical reaction Description automatically generated with medium confidence  A diagram of a graph Description automatically generated **Vmax** change is reduced -- **Km** is also reduced **Type of Inhibition** **Apparent** [**K**~**M**~]{.math.inline} **Apparent** [**V**~**max** ~]{.math.inline} ------------------------ -------------------------------------------- ----------------------------------------------- Competitive Increased Unchanged Non-competitive Unchanged Decreased Uncompetitive Decreased Decreased **Summary of the Effect of Enzyme Inhibition on the** [**K**~**M**~]{.math.inline} **and** [**V**~**max** ~]{.math.inline}  **The inhibitor Constant (**[**K**~**i**~]{.math.inline}**)** - A measure of the affinity of an inhibitor drug for an enzyme - [*K*~*i*~]{.math.inline} values are used to characterize and compare the effectiveness of inhibitors relative to [*K*~*M*~]{.math.inline} - Useful and important in evaluating the potential therapeutic value of inhibitor drugs for a given enzyme reaction - In general, the lower the [*K*~*i*~]{.math.inline} value, the tighter the binding, and hence the more effective an inhibitor is A graph of a slope and a slope Description automatically generated with medium confidence **Enzymes as Important Drug Targets** +-----------------+-----------------+-----------------+-----------------+ | **Disease** | **Enzyme** | **Drug** | **Mechanism** | +=================+=================+=================+=================+ | Hypertension | Angiotensin | Lisinopril | Inhibitor | | | Converting | | | | | Enzyme (ACE) | | | +-----------------+-----------------+-----------------+-----------------+ | Alzheimer's | Acetylcholinest | Donepezil | Inhibitor | | disease | erase | | | | | (AChE) | | | +-----------------+-----------------+-----------------+-----------------+ | Parkinson's | Dopamine | L-DOPA | Substrate | | disease | β-hydroxylase | | | +-----------------+-----------------+-----------------+-----------------+ | | Monoamine | Selegiline | Inhibitor | | | oxidase | | | | | | Moclobemide | | +-----------------+-----------------+-----------------+-----------------+ | Inflammation | Cyclooxygenase | NSAIDs, e.g. | Inhibitors | | | 2 (COX 2) | aspirin, | | | | | ibuprofen, | | | | | naproxen, | | | | | celecoxib | | +-----------------+-----------------+-----------------+-----------------+ | Gout | Xanthine | Allopurinol | Inhibitor | | | oxidase | | | +-----------------+-----------------+-----------------+-----------------+ | Cardiovascular | HMG CoA | Statins | Inhibitor | | disease | reductase | | | +-----------------+-----------------+-----------------+-----------------+