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Enzymes - Lecture detailed notes
Biochemistry/Lab (CHEM 3650)
Nova Southeastern University
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Enzymes Lecture notes (can be used for mcat review as well)
Enzymes as Biological Catalysts
Enzymes are biological catalysts. A catalyst does not impact the thermodynamics of a biological reaction, only help the reaction proceed at a faster rate.
Enzymes Classifications
The molecule upon which an enzyme acts are called substrates o Enzyme specificity: a given enzyme will only catalyze a single reaction or a class of reactions with these substrates Most enzymes have their named ending in the suffix – ase Oxidoreductases Catalyze redox reactions, usually with the help of cofactors to aid in electron carrying. o Electron donor is known as the reductant o Electron acceptor is known as the oxidant Enzymes with dehydrogenase or reductase Oxidase: enzymes with oxygen as their final electron acceptor Transferases Catalyze the movement of a functional group from one molecule to another Will be named with transferases in the name Kinases are also a part of this group o Catalyze the transfer of a phosphate group, generally from ATP, to another molecule.
Hydrolases Catalyzes the breaking of a compound into two molecules using the addition of water Are named only after their substrate o E. – phosphatase cleaves a phosphate group from another molecule o Peptidases (proteins), nucleases (nucleic acid), lipases (lipids) Lyases Catalyze the cleavage of a single molecule into two products Do not require water and do not act as oxidoreductases
Reverse reaction can also usually be catalyzed by lyase (two molecules synthesize one) o Known as synthases
Isomerases Catalyze the rearrangement of bonds within a molecule Can also be classified as oxidoreductases, transferases or lyases sometimes Catalyze reactions between stereoisomers and constitutional isomers Ligases Catalyze addition or synthesis reactions, generally between large similar molecules and often require ATP. Synthesis with smaller molecules is usually accomplished by lyases Most likely to be encountered in nucleic acid synthesis and repair
Impact on Activation Energy
Endergonic reaction: requires energy input (G>0) Exergonic Reactions: energy is given off (G<0) Catalysts exert their effect by lowering the activation energy of a reaction. o Make it easier for the substrate to reach the transition state
Mechanism of Enzyme Activity
Enzyme-Substrate Binding
Molecule upon which an enzyme acts is called the substrate. Together the two are known as an enzyme-substrate complex. Active Site: location within the enzyme where the substrate is held during the chemical reaction. o Assumes a defined spatial arrangement in the enzyme-substrate complex and this dictates the specificity of an enzyme for a molecule or group Two competing theories explain the interaction between enzymes and substrates. Lock and Key theory Suggests that the enzymes active site (lock) is already in the appropriate conformation for the substrate (key) to bind. Induced Fit Model More scientifically accepted theory
Michaelis-Menten Equation
Describes how the rate of reaction, v, depends on the concentration of both the enzyme [E] and the substrate [S], which forms product [P]. Concentration of enzyme is always kept constant o Velocity of the enzyme can be related to the substrate concentration: When this equation is equal to half of vmax, then Km = [S] Michaelis Constant, Km: is the substrate concentration at which half of the enzymes active sites are full o Used as a measure to compare enzymes since it measures the affinity of the enzyme to its substrate. The one with the higher Km has the lower affinity for its substrate since it requires a higher substrate concentration to be half-saturated
If [S] is below Km, then changes in substrate concentration will greatly affect the concentration rate.
Vmax: Represents the maximum enzyme velocity and is measured in moles of enzymes per second
Kcat: measures the number of substrate molecules converted to product, per enzyme molecule per second. o At low substrate concentrations, Km >>> [S], the Michaelis-Menton equation can be simplified to:
Catalytic Efficiency: ratio of kcat/Km indicates the efficiency of the enzyme.
Lineweaver-Burk Plots
Double reciprocal graph of the M-M equation. This graph yields a straight line Only real data is to the left of the y-axis (QUAD 1) X-intercept is equal to -1/KM Y-intercept is equal to 1/vmax
Cooperativity
Certain enzymes do not show classic hyperbola shape when M-M equation is graphed, instead show S-shaped sigmoidal due to cooperativity among substrate binding sites Cooperative enzymes have multiple subunits and multiple active sites o These subunits and enzymes may exist in one of two states Low-affinity Tense state (T)
High-affinity relaxed state (R) o Binding of substrate encourages the transition of other subunits from the T state to the R state, which increases the likelihood of substrate binding to other subunits. o Conversely, loss of a substrate can encourage other subunits to move from R state to T state. Often shown in regulatory enzymes inn pathways Quantified using Hill’s Coefficient o Hill’s Coefficient > 1: positively cooperative binding After one ligand is bound the affinity of the enzyme for further ligands increases o Hill’s Coefficient ><1: negatively cooperative binding After one ligand is bound the affinity of the enzyme for further ligands decreases o Hill’s Coefficient = 1: enzyme does not exhibit cooperative binding
Effects of Local Conditions on Enzyme Activity
Enzyme activity, Enzyme velocity, and enzyme rate are used interchangeably
Temperature
Enzyme-catalyzed reactions tend to double in velocity for every 10 degree increase in temperature until an optimum temperature is reached (37C/98F/310 K) o After optimum temperature is reached, activity falls of sharply if temp is increased Some enzymes are able to regain their function once cooled down.
pH
pH affects the ionization of the active sites A change in pH can also cause the denaturation of enzymes. Optimal pH is 7. Acidemia is when blood pH is less than 7. o Exceptions to this optimal level occur in the digestive tract Pepsin (stomach) works at a pH of 2 Pancreatic Enzymes work best in the small intestine at a pH of 8.
Salinity
Altering the concentration of salt can change enzyme activity in vitro Increasing levels of salt can disrupt hydrogen and ionic bonds which would cause a partial change in the conformation of the enzyme
Regulation of Enzyme Activity
Feedback Regulation
Feedback regulation: Enzymes are often subject to regulation by products further down a given metabolic pathway Feedforward regulation: enzymes regulated by intermediates that precede the enzyme in the pathway. Less common
Irreversible Inhibition
The active site is made unavailable for a prolonged period of time or is permanently altered. E. – Aspirin and other pain killing drugs are used to permanently disrupt the functioning of enzymes that help on creating pain-modulating products.
Regulated Enzymes
Allosteric Enzymes Have multiple sites: one active site and at least one allosteric site Allosteric Sites: regulate the availability of the active sites Allosteric Enzymes: alternate between an active and inactive form o Inactive form: cannot carry out the enzymatic reaction Molecules that bind to the allosteric site can be either allosteric inhibitors or allosteric activators. o Binding of either causes a conformational change in the protein An activator will result in a shift that makes the active site more available Inhibitor will make active sites less available Covalently Modified Enzymes Enzymes can be activated or deactivated by phosphorylation or dephosphorylation o Cannot determine whether it is activated or deactivated with experimentation Glycosylation is the covalent attachment of sugar moieties o Can tag an enzyme for transport within the cell or can modify protein activity and selectivity Zymogens Inactive form of potentially dangerous enzymes Contain a regulatory domain and a catalytic (active) domain. o Regulatory domain must either be altered or removed to expose the active site Have the suffix –ogen usually E. – trypsin has a zymogen form trypsinogen; Apoptotic enzyme (caspases)
Enzymes - Lecture detailed notes
Course: Biochemistry/Lab (CHEM 3650)
University: Nova Southeastern University
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