Enzymes 1 2020 PDF

Dr Mahani Mahadi © 2019, University of Cyberjaya. Please do not reproduce, redistribute or share without the prior express permission of the author.  Alhamdulillahirabbil 'alamiin. All praises be to Allah, the Lord of all the worlds. Blessings and Peace be upon Muhammad t...

Dr Mahani Mahadi © 2019, University of Cyberjaya. Please do not reproduce, redistribute or share without the prior express permission of the author.  Alhamdulillahirabbil 'alamiin. All praises be to Allah, the Lord of all the worlds. Blessings and Peace be upon Muhammad the Messenger of Allah, the Seal of the Prophets. O Allah, we thank You for the pleasure of having the knowledge that is blessed by You. We beseech You to bless our teachers and parents with Your Guidance. We pray for Your Guidance to become righteous students who are always close to You, and who also bring happiness to our teachers and parents. Amin Ya Rabbal 'alamiin. Enzymes Dr Mahani Mahadi  LEARNING OBJECTIVES At the end of this lecture, student should be able to Define enzyme List the basic properties of enzymes Classify enzymes Describe the factors affecting the enzyme action Explain Michaelis-Menten equation and describe the factor that influence the substrate concentration on the enzyme activities  Introduction Enzymes are biomolecules synthesized by all living cells. Enzymes act as biological catalyst (biocatalyst). CATALYST is a substance that increases the rate of chemical reaction (Velocity) (thus speed up the reaction from substrate  product)  They are protein in nature. There are some RNA molecules that act as enzyme. ie. Ribozymes – they serve to remove pieces of non- functional and functional portions mRNA called introns and exons Enzymes are thermolabile Enzyme has a high molecular weight (bigger than substrate)  General characteristics of enzymes 1. They are unchanged in the overall reaction, but may be temporarily modified during intermediate steps 2. They are effective in small amounts 3. They do not affect the equilibrium of a reversible chemical reaction. Enzyme affect only the kinetics (rate) not the thermodynamic properties of the reaction. Therefore, the same equilibrium will be reached with or without the enzyme, although it may not be reached in a reasonable time without the enzyme 4. They exhibit specificity in their ability to accelerate chemical reactions, but some may vary. Enzyme involved in the digestion are generally rather non- specific. Example: Proteases that hydrolyze proteins to peptides Many others are very specific in that they act only with a single substrate or with a very limited number of chemically similar compounds Example: Acetylcholinesterase is an enzyme that rather specifically catalyzes the hydrolysis of acetylcholine. 5. Many enzyme are localized in specific organelles within the cell. Such compartmentalization serves to isolate the reaction substrate or product from other competing reactions. in the cytosol in all organelles (nucleus, mitochondria, ribosome etc) In the membrane This provides a favorable environment for the reaction and organizes the thousands of enzyme present in the cells into purposeful pathway. 6. Enzyme activity can be regulated = increased or decreased, so that the rate or products formation responds to cellular need. 7. Enzyme molecules contain a special pocket or cleft called the active site. Because enzymes are protein, the active site contains amino acid side chains that participate in substrate binding and catalysis. The substrate binds to the active site of the enzyme, forming an enzyme-substrate (ES) complex. Binding is thought to cause conformational change in the enzyme (induced-fit) that allows catalysis. ES is then converted to an enzyme-product (EP) complex that subsequently dissociates to enzyme and product  Nature of enzymes The functional unit of the enzyme is known as holoenzyme which is often made up of apoenzyme (protein part) and a coenzyme (non-protein organic part). Some enzyme required helper molecules called cofactor. Cofactor divide into organic and inorganic substances. A coenzyme is an organic molecule that binds to an enzyme (transiently or permanently) and participates in catalysis. Many coenzymes, cofactors & prosthetic groups are derivatives of vitamin B.  Coenzymes serve as substrate shuttles. It is a soluble, organic molecule which promiscuously associates and dissociates with the various enzymes, e.g. NAD + and various dehydrogenases Prosthetic groups are tightly integrated into an enzyme’s structure. example FAD, is an organic molecule covalently bound to its partner enzyme Metal-activated enzymes: Some enzymes have increase activity due to presence of soluble metal ion co-factors , for example Mg++. Mg++ combines with ATP to provide the substrate for kinase reactions, e.g. hexokinase. Metalloenzymes: These are enzymes to which a metal is bound, e.g. alcohol dehydrogenase which is a zinc metalloenzyme. Zinc is bound to the sulphur atom of cysteine which is part of the reactive site. How are enzymes classified????  NOMENCLATURE OF ENZYMES Enzymes are characterized by the suffix –ase. Each enzyme can be identified in three different ways: (1) the recommended name, which is generally the historically grown name (e.g., hexokinase); (2) the “systematic name,” which describes the chemical function of an enzyme (e.g., ATP : D-hexose 6- phosphotransferase); and (3) The enzyme classification number (e.g., EC 2.7.1.1), which is based on the systematic naming system. Systematic Naming of Enzymes  There are (4) four steps: (1) An enzyme and a SUBSTRATE are in the same area. The substrate is the biological molecule that the enzyme will work on. (2) The enzyme grabs onto the substrate with a special area called How the ACTIVE SITE. The active site is a specially shaped area of the Enzymes enzyme that fits around the substrate. The active site is the keyhole of the lock. Work (3) A process called CATALYSIS happens. Catalysis is when the substrate is changed. It could be broken down or combined with another molecule to make something new. (4) The enzyme lets go. Big idea - When the enzyme lets go, it returns to normal, ready to do another reaction. The substrate is no longer the same. The substrate is now called the PRODUCT. 2 theories on how enzymes work  the shape of the active site matches the shape of its substrate molecules makes enzymes highly specific – each type of enzyme can catalyse only one type of reaction. If the shape of enzyme changes, its active site may no longer work. Induced Fit Hypothesis or Koshland model Because of the restriction nature of the lock-and-key model The important feature of this model is the flexibility of the region of the active site The active site does not possess a rigid, pre-formed structure on enzyme to fit the substrate However, the substrate during its binding induces conformational changes in the active site to attain the final catalytic shape and form This model explains: Why enzyme becomes inactive on denaturation Saturation kinetics Competitive inhibition Allosteric modulation  Catalytic activity of enzymes ENZYMES accelerate reactions at least a million times, by reducing the energy of activation. Before a chemical reaction can occur, the reacting molecules are required to gain a minimum amount of energy = energy of activation. An enzyme-catalyzed reaction is faster than an uncatalyzed reaction because it has a lower activation energy. Temperature and pH profoundly influence the activity of enzymes.  There are 2 ways to accelerate a chemical reaction FIRST: the reaction can be heated – increases the energy of the molecules and hence, increases the % of molecules with the required energy of activation. However, human body maintains a normal body temperature fairly constant. So increasing temperature might not work for human. SECOND: use of catalyst – they lower the energy of activation. Enzyme provides the reaction a different route. Enzyme catalysis of chemical reactions An enzyme-catalyzed reaction is faster than an uncatalyzed reaction because it has a lower activation energy. Temperature and pH profoundly influence the activity of enzymes. By lowering the activation energy, does enzyme increase the rate of the reaction? Does enzyme change the thermodynamic of the reaction? Does enzyme change the equilibrium (net product) of the reaction?  Factor affecting enzyme activity Concentration Concentration Effect of Effect of pH of enzyme of substrate temperature  Effect of pH pH affect the ionization of the active site The concentration of H+ affects reaction velocity in several ways. The catalytic process requires the enzyme and substrate to have specific chemical groups in either an ionized or non-ionized state to interact Extremes of pH can also lead to denaturation of the enzyme, because the structure of the catalytically active protein molecule depends on the ionic character of Pepsinthe amino optimum acid pH = side chains 2 ( digestive – stomach environment); Trypsin optimum pH = 6-7 ( pH in duodenum); Alkaline phosphatase = 8 ( pH in bone)  Effect of Temperature Increase of velocity (rate) with temperature is seen until a peak velocity is reached. This due to increased number of molecules having sufficient energy to pass over the energy barrier and form the products of the reaction Further elevation of the temperature results in a decrease in enzyme activity as a result of temperature-induced denaturation of the enzyme The optimum temperature for most human enzymes is between 35 and 40°C. Enzyme starts to denature above 40°C. Effect of [enzyme] As the concentration of the enzyme is increased, the velocity (rate) of the reaction proportionately increases. More enzyme and more active sites are made available for chemical reaction to occur thus increase the rate of the reaction  Effect of [substrate] In general, 1 enzyme molecule can bind with 1 molecule of substrate Thus the ratio of substrate molecule to enzyme molecule increase, the initial velocity (rate) increases with the proportion of [S] This will produce a linear graph and is termed the first-order reaction v (rate) proportional to [S] v = k [S] v = k [S]1  This proportionality decreases with increasing [S] depicting the saturation of enzyme for substrate binding – no free enzyme molecule that is available to bind with substrate molecule So no matter how much more substrate is added, the rate remains the same This will produce a plateau graph and is v (rate) proportional to [S] termed the zero-order reaction v = k [S] v = k [S]0 v =0  Other Factors Presence of cofactors and inhibitors: Enzymes often require cofactors, such as metal ions or coenzymes, to function optimally. The absence or limited availability of necessary cofactors can affect enzymatic activity. Additionally, the presence of inhibitors, which can be competitive or non-competitive, can interfere with enzyme-substrate interactions and reduce enzymatic activity. Enzyme stability: The stability of an enzyme, particularly its resistance to denaturation or degradation, is crucial for its overall activity. Various factors, such as temperature extremes, pH extremes, or exposure to chemicals, can affect the stability of enzymes and subsequently impact their activity.  The kinetic properties of enzymes Enzyme ( E) and substrate (S) combine to form unstable enzyme –substrate complex (ES) for the formation of product (P). K1, K2 and K3 represent the velocity constant for respective reaction Km or the Michaelis-Menten constant is defined as the [substrate] to produced half-maximum velocity in an enzyme catalysed reaction. This indicates that half of the enzyme molecules (50%) are bound to substrate molecules when the [substrate] equal to Km values. Km is representative for measuring the strength of ES complex.  The kinetic properties of enzymes The Michaelis–Menten equation is a rate equation for an enzyme-catalyzed reaction vmax is achieved when the substrate is saturated In an enzymatic reaction, Michaelis- Menten equation is represented by (Km) At half a vmax the [S] is equal to Km Or Km is [S] at half a vmax All enzymatic reaction happen at own rate constant (k) In the simplest case, an enzyme binds its substrate (in an enzyme–substrate complex) before converting it to product, so the overall reaction actually consists of first order and zero-order processes, each with a characteristic rate constant. Michaelis-Menten Equation  Km = [k-1 + k2] / k1 k-1 = the rate ES dissociation to E + S k2 = the rate ES dissociation to E + P k1 = the rate of formation of ES Km = [rate of dissociation] / [rate of formation] Km is a therefore a measure of affinity of an enzyme because High Km low affinity, less formation of ES Small Km high affinity, high formation of ES  Michaelis-Menten Equation v = Vmax [S] / Km + [S] if we know Vmax and Km of a particular enzyme we can actually calculate v at any [S] Lineweaver-Burk Plot Because of the difficulty of exactly determining Vmax from a hyperbolic graph, the Michaelis-Menten equation was transformed by Lineweaver and Burk into an equation for a straight line. The graph is 1/v versus 1/[S] v = Vmax [S] / Km + [S] 1/v = Km + [S] / Vmax [S] 1/v = Km / Vmax. 1 / [S] + [S] / Vmax [S] 1/v = Km / Vmax. 1 / [S] + 1 / Vmax (Lineweaver-Burk Equ) (y = mx + b) – linear plot Plot of Lineweaver-Burk graph Y intercept = 1 / Vmax  This form of the Michaelis-Menten equation is called the Lineweaver-Burk equation. For enzymes obeying the Michaelis-Menten relationship, a plot of 1/V0 versus 1/[S] yields a straight line (Fig. 1). This line has a slope of Km/Vmax, an intercept of 1/Vmax on the 1/V0 axis, and an intercept of −1/Km on the 1/[S] axis. The double-reciprocal presentation, also called a Lineweaver-Burk plot, has the great advantage of allowing a more accurate determination of Vmax Lineweaver-Burk Plot  For most enzymes, KM lies between 10-1 and 10- 7 M  What is kcat? The catalytic constant describes how quickly an enzyme can act k also called the turnover number. It is equivalent to cat the number of substrate molecules converted to product in a given unit of time on a single enzyme molecule when the enzyme is saturated with substrate. K is equal to Vmax/[Enzyme] cat k cat is the rate constant of the reaction when the enzyme is saturated with substrate (when [ES] < [E]T and v0 < Vmax).  Why is kcat/KM a better indicator of enzyme efficiency than kcat or KM alone? kcat/KM indicates catalytic efficiency The best way to compare the catalytic efficiencies of different enzymes or the turnover of different substrates by the same enzyme is to compare the ratio kcat/Km for the two reactions. This parameter, sometimes called the specificity constant, is the rate constant for the conversion of E + S to E + P. Kcat = Vmax/ [E]t  Determination of Km Worked example 1: An enzyme is discovered that catalyzes the chemical reaction. A team of motivated researchers sets out to study the enzyme, which they call happyase. They find that the k cat for happyase is 600 s−1 and carry out several additional experiments. When [Et] = 20 nM and [SAD] = 40 μM, the reaction velocity, V0, is 9.6 μM s−1. Calculate Km for the substrate SAD.  Determination of [S] In a separate happyase experiment using [Et] = 10 mM, the reaction velocity, V0, is measured as 3 μM s−1, what is the [S] used in this experiment? Solution: Using the same logic as in Worked Example 1, we see that the Vmax for this enzyme concentration is 6 μM s−1. Note that the V0 is exactly half of the Vmax. Recall that Km is by definition equal to the [S] at which V0 = ½Vmax. Thus, in this example, the [S] must be the same as the Km, or 10 μM. If V0 were anything other than ½Vmax, it would be simplest to use the expression,V0/Vmax = [S]/(Km + [S]) to solve for [S].  The happy couple’ Enzymes and reactants that are compatible are said to fit together like a ‘lock and key’. The happy couple shown in the bottom left of the illustration have corresponding shapes, and seem to be reacting well to each other in the warm environment. Thermostat Different enzymes have different optimum temperatures. Beyond their optimum temperature enzymes denature (breakdown). The thermostat in the illustration is set to a high temperature, and most of the enzymes seem uncomfortably hot. Arguments The shape of an enzyme’s active site determines which reactants it can bond with. In this illustration, incompatible enzyme–reactant pairs are shown either arguing or