Biochemistry Sheet 31 PDF 2024

31 Nadine Dababseh Zaynab Athamneh & Waqar Alfaqeer Dr Nafez Abutarboush 1 k₁: rate constant of formation the enzyme-substrate complex (ES). k₋₁: breakdown of ES into enzyme (E) and substrate (S) k₂: breakdown of ES into product( p) k₋₂: reformation of ES from E and the p...

31 Nadine Dababseh Zaynab Athamneh & Waqar Alfaqeer Dr Nafez Abutarboush 1 k₁: rate constant of formation the enzyme-substrate complex (ES). k₋₁: breakdown of ES into enzyme (E) and substrate (S) k₂: breakdown of ES into product( p) k₋₂: reformation of ES from E and the product(p ) Michealis-Menten put two assumptions to make final equation for reactions : 1- This reaction is irreversible; once you’re make a product you can’t get back to form reactants. 2- Steady state assumption ; the enzyme-substrate complex is in constant state, which means the rate of formation this complex equals the rate of degradation. For any enzyme in solution has two forms : Free ( E) and bound( ES) The enzyme ( E ) reach the ( Vmax) when E total( E+ES) are ES ( bound). The initial velocity of biochemical catalyze reactions to know velocity in which concentration of substrate equals the Vmax( inherint prperety of enzyme- not change- in y axis)that specific enzyme times S ( in x axis) the concentration to that velocity divided by 2 km+ S. The km = (K-1 +K2)/k1 -> the rate constant for degradation divide by rate constant of formation( its name (m ) to Michaelis.) The Michaelis constant (Km) This constant reflects the substrate concentration at which an enzyme operates at half(50%) of its maximum velocity (Vmax). It shows the enzyme's affinity for its substrate ( km it is an indicator for the affinity but not a real measure for the affinity , if we make it real we should remove K2 from equation that we call it KD). It is related to the rate of dissociation( degradation) of substrate to the enzyme substrate complex (ES). A quantitative description of the relationship between the rate of an enzyme catalyzed reaction (V0) & substrate concentration [S] A low Km indicates high substrate affinity, meaning the enzyme achieves half-maximal activity at a low substrate concentration. A high Km suggests low substrate affinity, requiring more substrate to reach the same level of activity. If more than one substrate was involved, each will have a different Km and Vmax.you should specify a graph for each one. 3  Relationship in Enzyme-Substrate Binding: Km represents the stability of the enzyme-substrate complex. (Reaction that’s one line) At the beginning of reaction the substrate concentration is very low so it can be neglected -> the reaction will be linear relation v=( Vmax[S])/Km. At the end of reaction the substrate concentration is very high and km is very low so it can be neglected it here so the reaction will be independent relation v = Vmax. If we put km the same value of substrate the equation will be v= Vmax /2 that the expression of definition of km. Dissociation Constant (Kd) Definition: A real measurement of the affinity between two molecules, such as an enzyme and its substrate or a receptor and a ligand. ( difficult to measure it that needs advanced measurements and just you study a complex unstable-transitional state- so we use km because it does not require a lot of money and easy) Represents: o The substrate concentration at which half of the enzyme binding sites are occupied. o Lower Kd → Higher binding affinity. Maximum Velocity (Vmax) Definition: The maximum rate of an enzymatic reaction when the enzyme is fully saturated with substrate. Indicates: o The enzyme's catalytic efficiency at its peak. o A higher Vmax means the enzyme can process substrate faster. What will happen to the Vmax and Km when the enzyme concentration is increased? V max increases( proportional & linear) and Km remains unchanged 4 Maximum Velocity (Vmax) Definition: The maximum rate of an enzymatic reaction when all enzyme molecules are saturated with substrate. (reveals the turnover number of an enzyme if the total concentration of active sites [E]T is known ) Represents: o The upper limit of the reaction rate for a given enzyme concentration. o Reflects how much product the enzyme can produce when it is working at full capacity. Turnover Number (Kcat) Definition: The concentration of substrate molecules converted into product by a single enzyme molecule per second when the enzyme is fully saturated with substrate.It is equal to the product of K2. Represents: o The intrinsic catalytic efficiency of an enzyme molecule. o A higher kcat indicates a faster enzyme. Turnover number; measures moles of substrate converted per unit time per moles of enzyme (min-1 or s-1)Turnover number = specific activity × molecular weight of enzyme 5 Relationship Between Vmax and kcat: Vmax describes the system-wide enzymatic activity, while kcat focuses on the efficiency of one enzyme molecule. Increasing enzyme concentration ([Etotal]) will increase Vmax but not kcat, since kcat is an intrinsic property of the enzyme. Example: a 10^-6 M solution of carbonic anhydrase catalyzes the formation of 0.6 M H2CO3 per second when it is fully saturated with substrate? Each catalyzed reaction takes place in a time equal to 1/k2, which is 1.7 μs for carbonic anhydrase The turnover numbers of most enzymes with their physiological substrates fall in the range from 1 to 10^4 per second Parameter Definition Factors that affect it Vmax Maximum reaction rate Enzyme concentration for the enzyme system Kcat Turnover rate of an Intrinsic property of enzyme enzyme Terms and Definitions Reaction rate; measures the concentration of substrate consumed (or product produced) per unit time (mol/{L.s} or M/s) Enzyme activity; measures the number of moles of substrate consumed (or product produced) per unit time (mol/s) ✓ Enzyme activity = rate of reaction × reaction volume Specific activity; measures moles of substrate converted per unit time per unit mass of enzyme (mol/{s.g}) * pay attention to the units * ✓ Specific activity = enzyme activity / actual mass of enzyme ✓ This is useful in determining enzyme purity after purification Turnover number; measures moles of substrate converted per unit time per moles of enzyme (min-1 or s-1) * the Same Kcat , K2* ✓ Turnover number = specific activity × molecular weight of enzyme Specificity and Efficiency (Kcat/Km) The spiecificity constant determines the relative rate of the reaction at low “S” kcat/KM (M^-1 min^-1) indicates: 6  Enzyme’s substrate specificity: the higher the ratio, the higher the specificity Enzyme’s catalytic efficiency: the higher the ratio, the more efficient the enzyme Parameter Definition Indication Km Substrate Enzyme substrate concentration for half of affinity Vmax Kd Dissociation constant Binding strength for enzyme substrate complex Vmax Maximum reaction rate Efficiency for an enzyme = enzyme activity / mass =2.675*10^-9/1.5*-6 =1.783*10-3 mol/(sg) = specific activity * Mw = 1.783*10-3 * 25*10^3 = 44.6 s^-1 7 Disadvantages of Michaelis-Menten equation and Lineweaver-Burk or double-reciprocal plot: Determining the Km from hyperbolic plots is not accurate since a large amount of substrate is required in order to reach Vmax The readings are not accurate because the equation is not linear This prevents the calculation of both Vmax & Km Lineweaver-Burk plot: A plot of 1/v0 versus 1/[S] (double- reciprocal plot), yields a straight line with an y-intercept of1/Vmax and a slope of KM/Vmax The intercept on the x-axis is -1/Km This is a linear equation However, this equation is not used because we can use softwares now …….…………………………………………………………………………… 8 ‫‪Km ≈ 8‬‬ ‫‪So the answer is E‬‬ ‫تمت كتابة هذا الشيت عن روح والدة زميلنا عمرو رائد من دفعة تيجان‬ ‫دعواتكم لها بالرحمة والمغفرة‬ ‫‪Thank you‬‬ ‫‪9‬‬