Enzymology
Enzyme inhibitor mechanisms
1) Decrease the enzyme's ability to bind substrate 2) Decrease the enzyme's catalytic activity 3) Both of the above
Methods to increase the Rate of the reaction:
1) Increase Temprature 2) increase Substrate Concentration 3) Lower the Activation Energy Catalysts and Enzyme lower the Activation Energy and increase Rate. Note: 1 & 2 = Increase reactant Kinetic Energy
Enzyme usually measured to diagnose Liver Damage
Alanine Aminotransferase
Heterotropic Effector
Allosteric Regulator: Positive Heterotropic Effector: Allosteric Activator Negative Heterotropic Effector: Allosteric Inhibitor
Apoenzyme vs. Holoenzyme
An inactive enzyme, without the cofactor is called an Apoenzyme, while the complete enzyme with cofactor is the Holoenzyme.
First Order Kinetics
Applyes to First order simple chmichal reactions like A--->B V₀=K[A] In Enzyme catalyzed reactions, When [S] is very small first order kinetics applies (V₀=K[A]) and when [S] is very large zero order kinetics applies (V₀=Vmax) and between these two limits (i.e. intermediate amount of S) Michaelis -Menten equation applies.
Michaelis-Menten kinetics
Approximately describes the kinetics of many enzymes. Relevant to situations where very simple kinetics can be assumed, (i.e. there is no intermediate or product inhibition, and there is no allostericity or cooperativity). More complex models exist for the cases where the assumptions of Michaelis-Menten kinetics are no longer appropriate. The Michaelis-Menten equation: The effect of Substrate Concentration on Reaction Kinetics
Reactant Kinetic Energy
Attaining activation energyn requires an increase in reactant kinetic energy: 1) Temprature 2) Substrate Concentration
Reversible vs Irreversible Inhibition
Both competitive and Non competitive inhibitions are reversible. Irreversible: Irreversible change in enzyme molocule and its function e.g. Aspirin (effect on COX is irreversible)
Non protein components which aid Enzymes
Cofactor: 1) Coenzyme 2) Prosthetic groups A cofactor is a non-protein chemical compound that is bound to a protein and is required for the protein's biological activity. These proteins are commonly enzymes, and cofactors can be considered "helper molecules" that assist in biochemical transformations. Cofactors can also be classified depending on how tightly they bind to an enzyme, with loosely-bound cofactors termed coenzymes and tightly-bound cofactors termed prosthetic groups. Important examples: Metals, Vit B Complex
Enzymes related to MI
Creatine Kinase Troponin T Lactate Dehydrogenase
Enzyme for converting Tripsinogen (Zymogen) to Tripsin (Enzyme)
Enteropeptidase
Enzyme
Enzymes are proteins that catalyze (i.e., increase the rates of) chemical reactions: Highly specific catalysts They just change the rate and can not alter the equlibrium. In enzymatic reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts them into different molecules, called the products
Allosteric enzymes
Enzymes that change their conformation upon binding of an effector. The kinetic properties of allosteric enzymes have been explained in terms of a conformational change between a low-activity, low-affinity "tense" or T state and a high-activity, high-affinity "relaxed" or R state. The kinetics of these enzymes doesn't follow Michaelis-Menten plot and their Velocity/Substrate curve is Sigmoid instead of Hyperbolic.
Zymogen
Inactive precursor of Enzyme: (Have Pro- befor or -gen after the name of the enzyme) * Angiotensinogen * Trypsinogen * Chymotrypsinogen * Pepsinogen * Most proteins in the coagulation system (Prothrombine, Plasminogen, Fibrinogen..) * Some of the proteins of the complement system * Caspases * Proelastase * Prolipase * Procarboxypolypeptidases Proinsuline is not a zymogen simply because Insuline is not enzyme.
Enzyme regulation
Increased Synthesis of the enzyme or Activation/Inactivation by: 1) Covalent modification: Phosphorylation, Dephosphorylation, Methylation, etc. 2) Proteolytic enzymes (Converting Zymogen to Enzyme or degradation of enzymes by proteases) 3) Allosteric modification (Could be a form of feedback regulation, if the end-product apply an allosteric change in the enzyme)
Isozyme
Isozymes (also known as isoenzymes) are enzymes that differ in amino acid sequence but catalyze the same chemical reaction. These enzymes usually display different kinetic parameters (e.g. different KM values), or different regulatory properties. The existence of isozymes permits the fine-tuning of metabolism to meet the particular needs of a given tissue or developmental stage
Michaelis Constant?
Km
Michaelis Constant
Km The substrate concentration when the Initial Velocity (Vo or Vi) is half of the Vmax
Competitive Enzyme Inhibition
Km Increased No effect on Vmax Both Inhibitor and Substrate compete for binding to the same site: Active Site Can be reversed with increased substrate concentration At last (With higher concentration of substrate) can reach the original Vmax because Enzyme activity and function still the same
Doubble Reciprocal Plot?
Lineweaver-Burk Plot
Non-competitive Enzyme Inhibition
No change in Km Vmax decreases Also called Allosteric inhibition. The site where the inhibitor binds is called Allosteric Site. (Separate from Active Site) Can not be reversed with increased substrate concentration Original Vmax could not be achieved because Conformational change has reduced the enzyme activity and function Can be a form of Feedback regulation
Competitive reversible inhibition
No change in Km and decrease in Vmax Hyperbolic curve of Michaelis-Menten plot shifts to the Right. Slope of Lineweaver-Burk plot Increases. (Rotates counterclockwise around 1/Vmax point on y-axis)
How Irreversible Inhibitors change the Km and Vmax
No change in Km and decrease in Vmax It's similar to Non-competitive form of Reversible Inhibition
Types of Enzymes
Oxidoreductase Hydrolase Lyase Transferase Isomerase Ligase
Allosteric Activator
Promote substrate binding (Increase cooperative binding of substrate) Shift the equlibrium of the 2 allosteric protein states from T to R Shift the Sigmoid Saturation curve or Binding curve to left
Non-Michaelis-Menten kinetics
Some enzymes produce a sigmoid V/[S] plot, which often indicates cooperative binding of substrate to the active site. This means that the binding of one substrate molecule affects the binding of subsequent substrate molecules. This behavior is most common in multimeric enzymes with several interacting active sites.
Induced fit model
Substrate binding > Conformational change of enzyme > Energy produced in favor of reaction
Enzyme Kinetics and Km
The reaction catalysed by an enzyme uses exactly the same reactants and produces exactly the same products as the uncatalysed reaction. Like other catalysts, enzymes do not alter the position of equilibrium between substrates and products. Enzyme-catalysed reactions display saturation kinetics: For a given enzyme concentration and for relatively low substrate concentrations, the reaction rate increases linearly with substrate concentration; the enzyme molecules are largely free to catalyze the reaction, and increasing substrate concentration means an increasing rate at which the enzyme and substrate molecules encounter one another. However, at relatively high substrate concentrations, the reaction rate asymptotically approaches the theoretical maximum; the enzyme active sites are almost all occupied and the reaction rate is determined by the intrinsic turnover rate of the enzyme. The substrate concentration midway between these two limiting cases is denoted by KM.
Thermodynamics vs Kinetics
Thermodynamics: The Energy states of a system, and the reaction Direction Concepts like ΔG, Exergonic, Endergonic, Reaction Direction and Equilibriubrium Kinetics: The Rate of the reaction and factors that affect the Rate Enzymes just alter the Kinetics (rate) of the reaction and have no effect on reaction equilibrium
Enzyme for converting Fibrinogen (Zymogen) to Fibrin (Enzyme)
Thrombin
In pure noncompetitive inhibition, the inhibitor binds with equal affinity to the free enzyme and to the enzyme-substrate (ES) complex. True or False?
True
At very high concentration of substrate the Michaelis-menten equation can be simplified to: ----
V₀=Vmax When [S] is too big, there is no difference between [S] and Km+[S] and the rate will be independent from [S]
Zero Order Kinetics
When [S] Saturates, Vmax achieved and The V₀ is no longer increase with increase of [S] V₀=Vmax
Km
[S] at 1/2Vmax another definition: E + S <---> ES ---> E + P Km = {Rate of ES breakdown to S or P}/{Rate of ES formation}
Mixed Enzyme Inhibition
a combination of two different types of reversible enzyme inhibition - competitive inhibition and uncompetitive inhibition. The term 'mixed' is used when the inhibitor can bind to either the free enzyme or the enzyme-substrate complex. In mixed inhibition, the inhibitor binds to a site different from the active site where the substrate binds. Mixed inhibition results in a decrease in the apparent affinity of the enzyme for the substrate (Increase in Km) and a decrease in the apparent maximum enzyme reaction rate (Decrease in Vmax)
