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73 Cards in this Set
- Front
- Back
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5 assumptions for michaelis-menton equation
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1) Catalysis the rate limiting step
2)Initial velocity (reverse reaction is 0) 3) Steady State 4) [S]>>>>>>> [ES] 5) [ET] = [EF] + [E-S] |
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3 necessary reasons to regulate enzyme activity
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1) Biological efficiency
2) Biological Flexibility 3) Control of competing reactions |
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4 features that make proteins good catalysts
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1) High Reaction Rates
2) Specificity: highly substrate and stereospecific, no side products 3) Mild reaction rates: can occur at biological pH, temp and pressure 4)Can be regulated: allowing for metabolic flexibility |
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Role of Ser195 in Chymotrypsin catalysis?
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attacks the carbonyl carbon in the peptide bond breaking the bond by covalent catalysis
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Role of Asp102 in chymotrypsin catalysis?
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The negative charge on Asp stabilizes the positive charge on His that forms during the cleavage of the peptide bond
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Role of His57 in chymotrypsin catalysis?
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Acts as a base to remove the H from serine. The proton is then donated to the amide N of the peptide bond during bond
cleavage. The His then abstracts a proton from water, during the hydrolysis of the acyl-enzyme intermediate. The proton is than restored to Ser195 to regenerate the enzyme as product is released. |
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3 roles of ATP
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1) Biosynthesis
2) Mechanical Work 3) active transport |
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NAD(P)+ transfers how many electrons?
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2 electron transfer
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FAD trasfers how many electrons?
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2 electron transfers, sequentially to become either FADH or FADH2
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Metals carry out how many electron transfers
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1 electron transfers
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2 routes of anaerobic glycolysis?
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1) Homlactate fermentations (lactate dehydrogenase)
2) Alcoholic Fermentation (alcohol dehydrogenase) |
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What are the three highly thermodynamically favorable, irreversible, regulated enzymes in glycolysis?
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1) Hexokinase
2) PFK 3) Pyruvate kinase 1,3,10 |
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Technique used to find regulation of enzyme amount?
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Microarray
ICAT 2D-SDS PAGE grow cells under two different conditions, differentially process (microarray/ICAT: different labels, 2Dgels: run on different gels), detect differences in protein levels |
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Technique used to find regulation of enzyme activity?
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Enzyme activity assays with activators and inhibitors
Run enzyme assays with and without compound, compare Vmax to see if and how enzyme activity changes |
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How does an active site form substrate specificity?
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Geometric (3 pt attachment)
Stereoselective |
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2 models of complentarity?
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Lock and Key
Induced |
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Principle of complementarity?
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1) Geometric complementarity
2) Chemical |
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What is in apoenzyme?
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An enzyme that needs a co-factor that does not have one
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What is a holoenzyme?
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Enzyme + Cofactor
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2 types of cofactors
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1) Metal Ions
2) Coenzymes |
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2 Types of Coenzymes
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1) Prosthetic groups. Regenerated within course of catalytic mechanism
2) Cosubstrates (NAD+). Regenerated by different enzyme. |
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Example of prosthetic group?
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Cytochrome
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Example of cosubstrate?
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NAD+ in alcohol and lactate dehydrogenase and glycolysis
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How do you identify amino acid composition in the active site?
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1) From the structure
2) Covalent modification of residues (inactivation of enzyme) |
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6 Mechanisms of Catalalysis
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Covalent
Acid-Base Metal Ion Electrostatic Proximity and Orientation Preferential Binding of Transition State |
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How does covalent catalysis lower activation energy?
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Not necessarily stabilizing transition state. Creating entirely new transition state.
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3 Characteristics that make metal ions good catalysts?
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1) Stabilize charges and not affected by pH
2) Involved in oxidation reduction reactions 3) Ionization of water |
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What is a metalloenzyme?
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Tightly bound metal
Essential in catalysis (Fe, Cu, Mn, Co) |
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What are metal activated enzymes?
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Loosely bound metals (Na, K, Ca)
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Rate of reactions depends on?
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Number of collisions
Orientation of molecules Reaction Pathway Energy of molecules |
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What is meant by proximity?
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The active site is very small and there is a high concentration of reactant within the active site.
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What makes up the catalytic triad in Serine proteases?
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Histidine
Aspartate Serine |
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Role of oxyanion hole in serine proteases?
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Stabilize transition state (not via electrostatic catalysis)
Example of preferential binding of transition state |
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What is the rate constant?
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proportional to the frequency of contact between substrates
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5 Assumptions of Michaelis-Menton Equation
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1) Catalysis is Rate-limiting (allows us to ignore binding)
2) Initial velocity (no product present, dont have to account for reverse reaction) 3) [S] is at steady state 4) [S] >>>> [ES] or [E] thus our concentration of S does not change at beginning of RXN 5) Et= Ef + ES |
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What is Km?
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The substrate concentration at which our initial velocity is equal to vmax/2. It represents affinity for substrate.
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What is Kcat?
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Our turn over number
How fast enzyme is working. Tells us number of substrate converted to product per unit time per enzyme |
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What is the slope of the double reciprocal equation?
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Km/Vmax
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How do negative effectors decrease enzyme activity?
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By either affecting substrate binding or catalysis
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3 Types of inhibitors (negative effectors)
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Complex
Uncompetitive Mixed- includes noncompetitive |
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Where do competitive inhibitors bind
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Active site
Effects binding not catalysis (Vmax does not change) |
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Uncompetitive inhibitors bind...
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ES in active site
Does not affect binding Does affect catalysis |
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Mixed inhibitors bind to...
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Regulatory Site inhibits both binding and catalysis
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Conditions affecting Enzyme Activity
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pH
Temp |
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2 Ways to have control of enzyme activity
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1) Enzyme Availability
2) Enzyme Catalytic Activity |
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3 Ways of controlling amount of enzyme amount
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1) Constitutive: always on
2) Inducible: Turned on in response to metabolic stimulus 3) Repressible: Turned off in response to metabolic stimulus |
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Example of inducible enzyme
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B galactosidase
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Example of repressible enzyme
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Enzymes of cholesterol biosynthesis
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Regulation of enzyme catalysis activity
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1) Covalent modification (Ex: Phosphorylation of PFK2)
2) Allosteric Regulation (non-covalent) |
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Example of irreversible covalent modification
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cleaving peptide
proteosomes |
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Rationale for Regulation
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Efficiency
Flexibility Competing Rxs/Branch points Avoid futile cycles |
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What is efficiency?
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Appropriate reactions running at right rates and right times
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Three types of complex feedback inhibition
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Cumulative
Concerted Isoenzymes |
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Energy Charge Equation
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(.5) (2ATP + ADP/ADP + ATP + AMP)
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LARGE EC will...
LOW EC will.. |
inhibit glycolysis
Activate glycolysis |
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E.C in the cell is ....
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.9
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Anabolism is a ..... process and allows for what three things
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Biosynthesis
Mechanical Work Active Transport |
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ATP hydrolysis is favorable because... (2 things)
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1) Charge repulsion relieved
2) Resonance |
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Source of ATP in chemotrophs
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Oxidation of organic compounds (carbs, lipids, proteins)
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What is oxidative phosphorylation?
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Used to synthesize ATP. Electrons are passed through electron transfer chain, cause H to pump into intermembrane space building up a gradient. H is pumped back out which converts ADP to ATP
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How do you detect stable isotopes?
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Mass Spec
NMR |
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What does proteomics tell us?
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Info about the different protein levels.
2 types: 2D-SDS-PAGE and ICAT (Isotope Coded Affinity Tag) |
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Summary of Stage I of glycolysis
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Glucose + 2ATP --> 2GA3P + 2ADP
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Summary of Stage II of glycolysis
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2GA3P + 2ADP + 2Pi --> 4 ATP + 2 pyruvate + 2NADH + 2H
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Summary of glycolysis
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Glucose + 2ADP + 2NAD+ + 2 Pi ---> 2 pyruvate + 2ATP + 2NADH + 2H+
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Results of conformation change of hexokinase
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1) Allows ATP to bind
2) Exclusion of Water 3) Proximity effect |
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What is the enzyme similar to hexokinase that is found only in the liver?
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Glucokinase
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What is our first committed step
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PFK
Phosphofructokinase |
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What is the rate determining reaction of glycolysis?
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The First committed step which is PFK
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Positive effectors of PFK
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Fructose-2,6-bisphosphate
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Negative effectors of PFK
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Citrate and ATP
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Positive effectors of pyruvate kinase?
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Fructose-1,6-bisphosphate
AMP and ADP |
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Negative effectors of pyruvate kinase?
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ATP, Acetyl-Coa, NADH
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