ITM 101 Biochemistry - Enzymes (G9 Version) - PDF

ITM 101 | BIOCHEMISTRY LESSON # 6- ENZYMES 1st YEAR | A.Y. 2023-2024 |SEPTEMBER 04, 2023 DR. RALPH CYLON JACINTO ACTIVATION ENERGY ENZYMES...

ITM 101 | BIOCHEMISTRY LESSON # 6- ENZYMES 1st YEAR | A.Y. 2023-2024 |SEPTEMBER 04, 2023 DR. RALPH CYLON JACINTO ACTIVATION ENERGY ENZYMES In chemical reactions there is an activation energy, it is the energy Objectives that must be overcome in order for the reaction to proceed Describe ∆G as a function of reaction rates. regardless if it is spontaneous or nonspontaneous. Define Keq and its significance. Discuss how enzymes catalyze reactions. Low activation energy → reaction proceeds rapidly Enumerate factors affecting enzyme activity High activations energy → slow reaction Explain the Michaelis-Menten equation & its significance. Discuss the different classes of enzymes. What is the main function of Enzymes? Enzymes are organic catalysts where their main function is to speed up metabolic functions in the body. A + B ⇌ P+ Q A + B to P+Q as to forward reaction and P + Q to A+B as to backward reaction. “⇌" symbol is used to denote an equilibrium; it means to say that the forward reaction is equivalent to backward reaction. There’s no net of formation of either A+B and P+Q; hence, they are in equilibrium. A+B → P+Q "→" symbol is used to denote a net forward reaction. The predominant is the forward reaction or the formation of P+Q. Note: There is still a backward reaction in this case, but the rate is not as fast as the forward reaction; hence, the predominant is P+Q. Note: If the reactants are at a lower free energy level than the FREE ENERGY products, it is endergonic. If the free energy level of the reactant is higher than the Free energy, ∆G – portion of the total energy in the system that is product, it is exergonic. available for work Question: ∆G = ∆H - T∆S In these two reactions, which among the two energies of their Where ∆G (Gibbs free) which is equal to ∆H (Enthalpy) total reactants are far from the activation energy? internal energy minus Temperature (T) multiplied by Entropy (∆S) Answer: ∆G = 0 → Equilibrium, no net change takes place Endergonic, because the initial energy of the reactant in exergonic ∆G ‹ 0 (negative) → “Exergonic”, spontaneous is already high enough that it only needs a small amount of energy ∆G › 0 (positive) → “Endergonic”, nonspontaneous to reach the activation energy as compared to the endergonic reaction. & The downhill movement of the ball is considered spontaneous because the starting ball would have a higher energy compared to the end ball. 101 INTRODUCTION TO MEDICINE | BIOCHEMISTRY AYURA 2027 1 ITM 101 | BIOCHEMISTRY LESSON # 6- ENZYMES 1st YEAR | A.Y. 2023-2024 |SEPTEMBER 04, 2023 DR. RALPH CYLON JACINTO EXERGONIC REACTIONS vs. ENDERGONIC REACTIONS One way of increasing this collision in between substrate molecules, would be to increase the movement of the molecules, EXERGONIC REACTIONS as a result, the temperature would increase. ○ Other term - Exothermic, that is, in exergonic reaction but the energy release is in the form of HEAT. The figure above shows the effect of increasing the substrate concentration. If the concentration of substrate increases, collisions between molecules increase, thereby facilitating the formation of the product. ENDERGONIC REACTIONS EQUILIBRIUM CONSTANT ○ Other term - Endothermic, that is, in an endergonic reaction but the energy released is in the form of HEAT. The equilibrium constant signifies the ratio of concentration of products and reactants. Furthermore, this will give an idea if the reaction is forward or backward, that is, whether it favors the formation of products or the formation of reactants. FORMULA: FACTORS AFFECTING REACTION RATES 1. TEMPERATURE 2. REACTANT CONCENTRATION 3. PRESENCE OF CATALYSTS (e.g Enzymes) If the temperature increases, the rate of reaction also increases. Note: If the temperature increases, the kinetic energy of the - Multiplied Products over multiplied Reactants. molecules increases, as well. Since the kinetic energy is - Bracket ([ ]) signifies molar concentration. the energy during motion, therefore, the molecules will move faster. Catalysts in general, speed up chemical reactions, the way Note: they speed up reactions can be illustrated by the figure In relation to circulation theory, in a chemical reaction the below: products will be formed if the molecules of the substrates collide. 101 INTRODUCTION TO MEDICINE | BIOCHEMISTRY AYURA 2027 2 ITM 101 | BIOCHEMISTRY LESSON # 6- ENZYMES 1st YEAR | A.Y. 2023-2024 |SEPTEMBER 04, 2023 DR. RALPH CYLON JACINTO - a). Enzymes would initially bind to the substrate. The process of substrate binding. - b). Forming enzyme-substrate complex. - c). Catalytic step where there is bond forming or breaking, forming the product. - d). After the product is formed, it will be detached from the enzyme itself. To illustrate the mechanism of enzymes, it actually lowers down the activation energy. As shown by the figure (The frogs must jump an amount of energy in order to below. become the product.) THEORIES ON ENZYME-SUBSTRATE BINDING - What enzymes will do is lower down the activation Substrate binding can be explained by theories which energy, to easily reach the formation of the product and include: thereby facilitate the reaction. ○ LOCK-AND-KEY MODEL The enzyme’s active site fits the Note: substrate perfectly. Enzymes are proteins that speed up chemical reactions When the enzymes approach the and NOT USED UP during the reactions they catalyze, which makes substrate, there is already an initial it unique. perfect fit between the substrate and the enzyme’s active site. For enzymes to catalyze, two (2) steps are involved: This explains why enzymes are specific when it comes to the substrate. 1. They must bind to the substrate. If this enzyme is acting on a 2. They facilitate catalysis (catalytic step). During this step, protein, any other molecule this is where bonds are being formed or broken, that isn’t a protein (e.g. producing products. carbohydrate) would not fit the active site and therefore would not be acted upon by the enzyme. Example. Note: If this theory is followed, it could mean that enzymes are rigid, which is not the case for proteins. Furthermore, there are enzymes that can bind to several substrates. Example, Hexoses 101 INTRODUCTION TO MEDICINE | BIOCHEMISTRY AYURA 2027 3 ITM 101 | BIOCHEMISTRY LESSON # 6- ENZYMES 1st YEAR | A.Y. 2023-2024 |SEPTEMBER 04, 2023 DR. RALPH CYLON JACINTO which can bind to glucose, galactose and fructose. Therefore, this COENZYME is not the best model to explain substrate binding. Big, Non-protein. Organic molecules that required the same ○ INDUCED FIT MODEL enzyme to activate them. Initially, there is no perfect fit between Participates in binding to the substrate the active site and the substrate, bind s to the active site to prepare the active site however, if the substrate is in close for substrate binding proximity to the active site of the By them don't have any catalytic activity enzyme, the enzyme will change its Help catalyze reactions by: conformation-modification will happen ○ donate/ accept electrons in order to fit the substrate. ○ transfer group When the enzyme approaches the ○ form/break covalent bonds substrate, the substrate induces a ○ Provide functional groups change in the active site so note : function similar to co-factor but not metallic ions instead complementarity will be achieved - they are organic molecules conformational changes - eventually achieving a perfect fit. COMMON COENZYMES Modification of the lock-and-key model. LIPOIC ACID ○ Decarboxylate alpha-keto acid NOMENCLATURES NAD/NADP,FAD ○ Redox reaction 1. Active Site ○ Transfer of electron - Portion of enzymes which folds to precisely fit the ○ Dehydrogenation contours of a substrate via weak electrostatic interactions ○ Transfer of H+ and facilitates bond reactivity. (the portion where CoASH enzymes directly bind to the substrate). ○ Kreb’s cycle ○ Beta oxidation VITAMINS ○ The body needs vitamins such as ascorbic acid, cyanocobalamin and folic acid ○ By themselves, they do not provide energy but instead help unlock energy by acting as coenzymes for some metabolic reactions - Active sites are specific. That is, it can only bind certain HOLOENZYME types of substances. The entire enzyme together with all the necessary - Ex. An enzyme that can specifically bind to cofactor plus the protein portion. glucose. In which, it can not bind to other The complete enzyme monosaccharides except glucose. 2. Simple enzymes - Only made up of protein. 3. Co-factors - Small, in-organic, metal ions that are required of some enzymes to activate them. - Usually located near the active site. APOENZYME - Helps in binding to the substrate. Simple enzyme - Example: Mg++ stabilizes the carbonyl oxygen in Taken out cofactor or coenzyme, only left with the protein the phosphoenol pyruvate to allow the enzyme portion enolase to act on it; Mg++ participates in the catalytic activity. - Other examples: Cu, Mn, Fe, etc. - Important in order for enzymes to facilitate the activity. 101 INTRODUCTION TO MEDICINE | BIOCHEMISTRY AYURA 2027 4 ITM 101 | BIOCHEMISTRY LESSON # 6- ENZYMES 1st YEAR | A.Y. 2023-2024 |SEPTEMBER 04, 2023 DR. RALPH CYLON JACINTO PROSTHETIC GROUPS There are certain cofactor or coenzymes that are difficult to separate from the portion of the enzyme because they are covalently bound to the enzyme example : Heme ○ The structure shown below here that is present not just in hemoglobin but in other enzyme like catalase 3. Substrate concentration Initially, if substrate concentration increases, the enzymatic activity will also increases (directly proportional) However, once reached the maximum velocity, the enzymatic activity will no longer increase because the enzymes are already occupied (saturated) ○ Letter c shows us that the enzymes are already occupied. It can be graphs using the Michaelis-Menten FACTORS AFFECTING ENZYMES equation. 1. Temperature Enzymes are protein and extremes temperature can denatured enzymes note :graph shows us the temperature vs the enzymatic activity, as the temperature increases initially, the enzymatic activity increases but up to a certain point only, this point termed as optimal temperature The enzymatic activity in the human body is about 40. More than that the enzymatic activity starts to inactivated or denatured Michaelis-Menten Curve 2. pH value Also have an optimum pH However, each enzymes have their own specific optimum pH example , pepsin optimum pH is around pH 1 (acidic) and enzyme in small intestine optimum Initially, as you increase the substrate pH is around pH 8 concentration it is directly proportional to the reaction rate, up to a certain point. When the enzyme is saturated, further increasing the substrate concentration leads to slower reaction rate. 101 INTRODUCTION TO MEDICINE | BIOCHEMISTRY AYURA 2027 5 ITM 101 | BIOCHEMISTRY LESSON # 6- ENZYMES 1st YEAR | A.Y. 2023-2024 |SEPTEMBER 04, 2023 DR. RALPH CYLON JACINTO Michaelis-Menten Curve Plot the reaction of the substrate concentration against the reaction rate. What is obtained is a hyperbolic curve. It is a combination of the zero order and the first order. Initially, it follows the first order reaction, mixed, then zero order kinetics becoming independent of the reactant concentration upon reaching the Vmax. Important Parameters in Michaelis-Menten Equation: Vmax ○ Maximum velocity of the reaction ○ Never be reach by the chemical reaction ○ ½ Vmax or 50% maximum velocity [S] - Substrate concentration Vi - reaction rate Km ○ Michaelis constant ○ To determine the Km, get 50% of Vmax plot it against the x-axis or the substrate concentration. ○ A point/substrate concentration wherein the reaction is at 50% of the maximum velocity. ○ If the Km on an enzyme is high, it is low affinity to the substrate. Because high substrate concentration results in low saturation. Zero order Reaction: Even if we increase the reactant concentration, the reaction rate stays the same, it doesn’t change, it is constant. There are two available enzymes one is high Km and one The reaction rate is independent on the reactant has low KM. among these two enzyme a it’s smaller easily concentration. Even if there are a lot of reactants, the saturated because of its high affinity. reaction rate remains constant. The Vmax of enzyme A is very much slower compared to Zero reaction rate enzyme B Independent of the reactant concentration the reaction rate is constant regardless of how high or low the reactant concentration is. Fixed reaction rate of zero reaction rate. First order reaction Even if we increase the reactant concentration, there is a corresponding increase in the reaction rate Direct, proportional relationship between reactant concentration and enzyme reaction rate (if we the increased reaction concentration the reaction rate increases) The reaction rate is dependent on the reactant concentration. the more the reactants the more faster the ( a converted graph of michaelis menten curve) reaction rate a double reciprocal (fraction) of each point of michaelis menten curve making it into linear function called Lineweaver-Burke plot. Algebra: Y=mx + b 101 INTRODUCTION TO MEDICINE | BIOCHEMISTRY AYURA 2027 6 ITM 101 | BIOCHEMISTRY LESSON # 6- ENZYMES 1st YEAR | A.Y. 2023-2024 |SEPTEMBER 04, 2023 DR. RALPH CYLON JACINTO m= slope x= value of x in x-axis y= value of Y in Y-axis In lineWeaver Burk plot Y= reciprocal of initial velocity Slope= km/Vmax x= 1/[S] b= 1/Vmax X= -1/km or negative reciprocal of km Important note: lineweaver Burke plot gives an idea of the different mechanisms of enzyme inhibitors. Enzyme inhibitors Competitive inhibitor noncompetitive inhibitor Noncompetitive inhibitor Uncompetitive inhibitor Affinity is not affected Note: Each enzyme inhibitors have varying effect on each enzyme. -1 / Km is constant but Vmax is decreased It lowers down the overall reaction rate but it will not affect the affinity of the substrate to the enzyme. They are not competing with the same binding site. the substrate binds to the active site, the inhibitor binds to another site called the allosteric site- site other than the active site. The inhibitor can bind to the free enzyme, it can also bind to the enzyme that is already bound to the substrate. inhibitors never bind to the active site. both EI and EIS complexes are enzymatically inactive. Allosteric side causes a conformational change to the active site inhibiting the substrate from binding to the active site causing the reaction rate to decrease. Competitive inhibitor One over Vmax is not affected. Constant. (down in the point of intersection between the two lines) -1 / Km increased (from negative 1/3 to negative 1/4) Competitive inhibitor Decrease the affinity of the enzyme to the substrate without affecting the overall maximum velocity of the reaction. The substrate and the inhibitor compete for the same binding site which is the active site. If it is the substrate that binds to the enzyme, well and good. if it is the inhibitor that binds the enzyme, this turns the enzyme inactive. this is a reversible type of inhibition. The Uncompetitive inhibitor inhibitors often resemble the substrate structurally, they Lowers down the Vmax of the whole reaction and, at the are said to molecular analogous. same time, it would increase the affinity of this substrate causes decrease in affinity to the enzyme. Reversible inhibitor because when further increase the Vmax decreased amount of substrate the inhibitor can be bumped off from Km decreased the active site. Occurs when they inhibit binds only to the ES complex to form the EIS complex Inhibition is irreversible 101 INTRODUCTION TO MEDICINE | BIOCHEMISTRY AYURA 2027 7 ITM 101 | BIOCHEMISTRY LESSON # 6- ENZYMES 1st YEAR | A.Y. 2023-2024 |SEPTEMBER 04, 2023 DR. RALPH CYLON JACINTO 101 INTRODUCTION TO MEDICINE | BIOCHEMISTRY AYURA 2027 8