Enzymes Flashcards
Catalysts
- do not impact thermodynamics of a reaction
- enthalpy (H) of reaction and equilibrium do not change
- help reaction proceed at faster rate by lowering activation energy or changing the reaction mechanism
List key features of enzymes
- lower activation energy (Ea) so forward and reverse rxns occur more often
- increase rate of the rxn
- no change in equilibrium constant
- appear in both reactants and products b/c not consumed in the rxn
- pH and temperature sensitve
- no change in Gibbs Free Energy of rxn
- specific for a particular rxn or class of rxns
What are the 6 different enzyme classifications?
- LIL HOT*
- Lyase
- Isomerase
- Ligase
- Hydrolase
- Oxidoreductase
- Transferase
Oxidoreductase
- catalyzes oxidation-reduction rxns
- catalyze the transfer of electrons between biological molecules that contain elements like C, H, and O
- often have cofactor like NAD+ or NADP+
- electron donor is the reductant
- electron acceptor is the oxidant
- reducing a molecule means fewer bonds to oxygen and more bonds to hydrogen: A + H AH
- oxidizing a molecule means more bonds to oxygen and less bonds to hydrogen: A + O AO
- enzymes that have oxygen as the final electron acceptor include “oxidase” in their name
- Ex. oxidases, reductases, peroxidases, dehydrogenases
Transferase
- catalyze movement of a functional group from one molecule to another
- kinases are an example of this and function in catalyzing the transfer of a phosphate group to another molecule
- Ex. polymerases (shift nucleotides into growing chains of DNA or RNA), transaminases, methyltransferases, hexokinase
Hydrolase
- catalyze the breaking of a compound into two molecules using the addition of H2O
- AB + H20 –> AOH + BH
- Ex. phosphatase (cleaves phosphate group in peptide bond), peptidase (breaks down proteins), nuclease (break down nucleic acids/DNA/RNA), lipase (break down lipids), protease (breaks peptide bonds within proteins), esterase (breaks esters often in lipids)
Lyase
-catalyze the cleavage of a single molecule into two products without the use of water
-also involved in cleavage of bonds (can cleave dbs to make single bonds)
-often form rings or multiple bonds to reform octets
- XABY –> AB (ring form) + XY
- A = B + XY
-common names: decarboxylase, lyase, synthase
-Ex. use of enzyme aldolase:
Fructose 1.6-bisphosphate DHAP + Glyc. 3-phosphate
-Ex. glycogen phosphorylase
Isomerase
- catalyze the rearrangement of bonds within a molecule – intramolecular
- have same chemical formulas with different connectivity
- AB BA
- catalyze reactions between stereoisomers and constitutional isomers
- some can also be classified as oxidoreductases, transferases, or lyases
- common names: mutase, racemase
- Ex. triose phosphate isomerase, aconitase
Ligase
- catalyze addition and synthesis reactions, generally between large similar molecules and often require ATP
- X + Y + ATP –> XY + ADP +Pi
- usually involved in nucleic acid synthesis or DNA synthesis/repair
- the only enzyme class that absolutely needs ATP to function
- common enzymes in this class: synthetase, carboxylase
- Ex. DNA Ligase, carbonic anhydrase
Endergonic Reactions
requires energy input (delta G > 0)
Exergonic Reactions
energy is given off (delta G < 0)
What are the mechanisms of enzyme activity that lead to decreased activation engergy?
- Transition State Stabilization: makes transition state exist longer, dissipation of torsional strain and favorable bond formation, inductive effects of the active site residues
- Microenvironment Adjustments: keeps H2O away form molecule, adjusts local environment’s pH
- Substrate Proximity Adjustments: increases frequency of collisions
- Transient Covalent Bonding: substrates are vulnerable to nucleophilic attack so briefly contact active site residues
- Reactant Destabilization: creation of torsional strain or hydrophobic-hydrophilic interactions that make the rxn favorable because molecules are in the wrong state
What stabilizes the spatial arrangement within the active site of an enzyme when a substrate is present?
- hydrogen bonding
- ionic interactions
- transient covalent bonds
Lock and Key Theory
- describes enzyme-substrate binding
- enzyme’s active site (lock) is already in correct configuration for substrate (key)
- no alteration of tertiary or quaternary structure is necessary upon binding of substrate
- problems with model: competitive inhibition (more than one key for a given lock), promiscuous reactivity, reverse catalysis
Induced Fit Model
- describes enzyme-substrate binding
- active site of enzyme molds itself around substrate only when substrate is present
- tertiary or quaternary structure is necessary upon binding of substrate
- more accurate model of the two
Coenzymes
- extrinsic ORGANIC molecules that are necessary for protein function
- many are adenine or vitamin derived
Prosthetic Groups
- tightly bound cofactors or coenzymes that are necessary for enzyme function
- Ex. cysteine residue on heme C
Ribozymes
biological catalysts that are composed of RNA instead of polypeptides
What 2 vitamins are soluble in water?
B, C
What 4 vitamins are soluble in fat?
K, E, D, A
Cofactors
- INORGANIC molecules that are necessary for protein function
- usually free metal ions but can be polyatomic
Haloenzymes
enzymes that have all necessary cofactors and coenzymes present
Apoenzyme
enzymes that do not have all cofactors and coenzymes present
The site where a protein binds essential cofactors is most likely to be characterized by:
an excess of negative charge which is because most cofactors are metal cations
What amino acid is found in the active site of chymotrypsin and what molecule does it bind?
Serine acts as the active site and it binds phynylalanine
What amino acid is found in the active site of trypsin and what molecule does it bind?
Aspartate acts as the active site and it binds lysine
Saturation Kinetics
- point at which an enzyme can’t go any faster
- enzyme is working at max velocity (Vmax) which can only be increased by increasing the enzyme concentration
Michaelis-Menten Constant (Km)
- inherent measure of the affinity a substance has for an enzyme
- used to compare two enzymes to see which would perform better
- substrate concentration such that V=1/2Vmax – half of all enzyme active sites are full
What do high values of Km represent?
- low affinity enzyme substrate complexes (because it requires a higher substrate concentration to be 1/2 saturated)
- slow enzyme rxns (decreased Vmax)
What equation can be used if the reaction rate is equal to half of Vmax?
Km = [S]
What does a low Km value represent?
high affinity for the substrate (low substrate concentration required for 1/2 enzyme saturation)
What equation can be used when [S]»_space;> Km (zeroth order region)?
V = Vmax
What happens to the reaction rate when [S] > Km ?
reaction rate increases much slower as it approaches Vmax
What equation can be used when [S] «< Km (first order region)?
V = (Vmax x [S] ) / Km
Catalytic Efficiency
- Kcat / Km
- large Kcat (high turnover) or small Km (high substrate affinity) results in a higher efficiency – more efficient enzyme
- “perfect” value of this is between 10^8 and 10^9
Lineweaver-Burk Plots
- double reciprocal graph of Michaelis-Menten equation
- plot is used to determine the type of inhibitor that an enzyme is experiencing because Vmax and Km can be directly compared
- -1/Km is the intercept of the line with the x-axis
- 1/Vmax i the intercept of the line with the y-axis
Cooperativity
- graph shows sigmoidal (S-shaped) kinetics owing to cooperativity among substrate binding sites
- these types of enzymes have multiple subunits and active sites
- enzymes exist in two stages: low-affinity tense state (T) or high-affinity relaxed state (R)
- Ex. hemoglobin
What encourages transition from T state to R state?
substrate binding because it increases the likelihood of substrate binding by other subunits
Hill’s Coefficient
numerical value that quantifies cooperativity
Hill’s Coefficient > 1 :
positive binding is occurring –> after one ligand is bound the affinity of enzyme for more ligand binding is increased
Hill’s Coefficient < 1 :
negative binding is occurring –> after one ligand is bound the affinity of enzyme for more ligands decreases
Hill’s Coefficient = 1 :
enzyme doesn’t exhibit cooperative binding
How does cooperativity work in hemoglobin?
binding of first O2 to Fe increases polarity and makes other Fe more available to bind O2
What local conditions effect enzyme activity?
- temperature
- pH
- salinity
What are the effects of temperature on enzyme activity?
- enzyme rxns double in velocity every 10 degrees Celsius until optimum temperature is reached
- enzyme denatures at high temperatures past the optimum temperature
What are the effects of pH on enzyme activity?
- impact on ionization of active site and can lead to enzyme denaturation
- optimal enzyme pH in human blood: 7.4 (if pH is less than this then termed acidemia)
What is the ideal pH of a gastric enzyme?
2
What is the ideal pH of a pancreatic enzyme?
8.5
What are the effects of salinity on enzyme activity?
increasing levels of salt in vitro (in lab) can disrupt H-bonds and ionic bonds causing a change in enzyme conformation and denaturation
Feedback Regulation
process by which enzymes are subject to regulation by products further down a given metabolic pathway
Feed-Forward Regulation
when enzymes are regulated by intermediates that precede the enzyme in the pathway
Feedback Inhibition
regulatory mechanism where catalytic activity of enzyme is inhibited by high levels of product later in the same pathway
What are the 4 types of reversible inhibition?
- competitive
- noncompetitive
- mixed
- uncompetitive
Competitive Inhibition
- inhibitor is similar to the substrate and binds at the active site
- overcome by adding more substrate
- Vmax is unchanged
- Km increases
Noncompetitive Inhibition
- inhibitor binds to allosteric site which induces a change in enzyme conformation
- inhibitors bind with equal affinity to enzyme and ES complex
- Vmax is decreased
- Km is unchanged
Allosteric Sites
non-catalytic regions of the enzyme that bind regulators
Mixed Inhibition
- inhibitor binds at allosteric site
- inhibitor binds with unequal affinity to enzyme and ES complex
- Vmax is decreased
- Km increases (decreased affinity) if inhibitor preferentially binds to enzyme
- Km decreases (increased affinity) if inhibitor preferentially binds to the ES complex
Uncompetitive Inhibition
- inhibitor binds to allosteric site after ES complex is formed
- inhibitor only binds to ES complex
- both Vmax and Km decrease
Irreversible Inhibition
-alters enzyme in such a way that the active site is unavailable for a prolonged duration or permanently
Allosteric Enzymes
- multiple binding sites (active site + regulatory sites)
- alternate between active and inactive form
- bind allosteric activators or inhibitors which both cause conformational shift in protein
Covalently Modified Enzymes
- activated by phosphorylation
- deactivated by dephosphorylation
- glycosylation: covalent attachment of sugar moieties; tag an enzyme for in-cell transport or modify protein activity and selectivity
Zymogens
- secreted in inactive form and are activated by cleavage
- contain a catalytic (active) domain and a regulatory domain
- have suffix -ogen
What are intrinsic parameters of an enzyme?
- parameters that “belong” to the enzyme and won’t change even if the concentration of enzyme changes – NOT dependent on [E]
- includes: Km, Kcat, catalytic efficiency
What are extrinsic parameters of an enzyme?
- parameters that can change as enzyme concentration changes – dependent on [E]
- includes: Vmax
Ordered Sequential Binding
- substrates must bind in correct order for product(s) to form
- must bind substrates before product can be made
Randon Sequential Binding
-all substrates must be bound before product(s) can be formed, BUT substrates can bind in any order and products can be released in any order
Ping Pong Binding
- release some products before you bind all substrates
- only forms binary complex (enzyme + substrate), NEVER forms tertiary complex (enzyme + substrate + inhibitor + …)
- Ex) Aspartate + alpha-KG OAA + Glutamate
