Enzymes and Kinetics Flashcards
histone acetyltransferases:
add acetyl groups, makes DNA more accessible for transcription
- histone deacetyltransferases reduce transcription
Km
Km is the concentration of substrate which permits the enzyme to achieve half Vmax.
An enzyme with a high Km has a low affinity for its substrate, and requires a greater concentration of substrate to achieve Vmax.”
Mixed Inhibitors
Can bind to free enzyme (no substrate bound) of enzyme-substrate complex
Decreases Vmax
Km depends on binding preference, increases when bound to free enzyme
Proteases
Trypsin
Pepsin
Proteases cleave peptide bonds
trypsinogen cleaved by enteropeptidase (activated by CCK) into active form, trypsin
- trypsin: cleaves peptide bonds adjacent to lysine and arginine
Pepsinogen cleaved by stomach acid into pepsin; cleaves bonds between hydrophobic and aromatic aminos
Kinase vs. Phosphorylase
Kinase is not involved in breaking bonds in the substrate during the addition of phosphate groups whereas phosphorylase breaks the bond between the substrate and the monomer by adding a phosphate group.
Glycosyltransferases
Glycosidase
Glycosyltransferases create new glycosidic bonds
- glycogen synthase in the liver
Glycosidase enzyme breaks glycosidic bonds via acid-catalyzed hydrolysis reaction
- Beta-galactosidase hydrolyzes glycosidic bond of lactose to liberate glucose and galactose
**Always involve anomeric carbon (carbonyl carbon) of at least one sugar
peptide hormones
Chains of amino acids, large and polar
- Can’t diffuse into cell, must interact via membrane receptors and secondary messengers (QUICK ONSET< SHORT LASTING)
Phospholipids can move horizontally but not vertically
- need enzymes to catalyze movement:
Flippase: moves phospholipids from external side to internal side
Floppase: opposite
Scramblaser: bidirectional
transmembrane ATPases catalyze
hydrolysis of ATP
ex. Sodium Potassium pump
Na+K+ ATPase
ex. myosin
Oxidoreductases
catalyze oxidation reduction reactions
- oxidases
- reductases
- dehydrogenases
Hydrolases
General term that catalyze a hydrolytic cleavage
Hydrolysis is the chemical breakdown of a compound due to reaction with water.
- Nucleases and proteases
Nucleases
Break down nucleic acids by hydrolyzing bonds between nucleotides
Synthases
Synthesize molecules in anabolic reactions by condensing two smaller molecules together
Isomerases
Catalyze the rearrangement of bonds within a single molecule
Polymerases
Catalyze polymerization reactions such as the synthesis of DNA and RNA
Phosphatases
Catalyze the hydrolytic removal of a phosphate group from a molecule
Thrombin
an enzyme in blood plasma which causes the clotting of blood by converting fibrinogen to fibrin.
Lysozyme
It functions as an antimicrobial agent by cleaving the peptidoglycan component of bacterial cell walls, which leads to cell death.
Transferases
Transfer functional group between molecules
Ribosomal rRNA
ribozymal component of ribosomes, catalyzes formation of peptide bonds
Cyclins
Present at each stage of cell cycle (specialize), levels are cyclical and spike when its time to use them
when at high levels, bind and activate cyclin dependent kinases which phosphorylate and activate proteins which promote activity of a certain phase
Prosthetic Groups
Coenzymes tightly or covalently bonded to their enzymes
ex. Heme attached to O2 transporters like hemoglobin or myoglobin
Substrate
Substance an enzyme acts on
Committed step of reaction
Energetically unfavorable, highly regulated
Orders of reactions
Zero order reactions: unaffected by changes in reactant concentrations
- ex. enzyme catalyzed reactions in which enzyme is saturated.
- reactant concentrations far exceed available active site
First order: radioactive decay or SN1 reactions, rate only depends on one reactant
Second order: physical collisions between two reactant molecules
Rate Law
In one step elementary rxns, coefficients of reactants are their orders
Rate= k [A]a[B]b
Multistep: using initial rates, find where conc of [A] changes and the other [B] stays the same
a and b are the rate orders of each reactant
rate1/rate2 = (conc1/conc2)x
Rate of 1 = no change
apoenzymes
Enzymes without cofactors needed to function properly
Zymogen
Inactive precursor molecule
allosteric regulation
allosteric regulation (or allosteric control) is the regulation of an enzyme by binding an effector molecule at a site other than the enzyme’s active site.
competitive inhibition
Only inhibiton at enzyme active site, only one that inhibits binding of substrate
- increase Km (lower affinity)
- no change in Vmax
Line weaver burk plots
*Y-AXIS moves in OPPOSITE direction (Vmax)
- if reduced, moves up
X-axis moves in correct direction
Noncompetitive inhibition
Binds on E-S complex, doesn’t inhibit binding of substrate and therefore doesn’t change Km, (noncompetitive)
- Decrease in Vmax (for all but competitive, which stays the same)
uncompetitive inhibition
Binds to ES complex or enzyme and decreases Km
- Decreases Vmax
Enzyme inhibition regular graph
Restriction enzymes can only recognize
Palindromic sequences
Isoelectric point
pH of a solution at which the net charge of a protein becomes zero.
How can enzymes be used to form a single stereoisomer?
An enzyme is a chiral catalyst capable of preferentially forming one enantiomer because a lower energy chiral transition state leads to the preferred product
What enzyme creates glycosidic bonds, what enzyme breaks them down
Glycosyltransferases create new glycosidic bonds
- glycogen synthase in the liver
Glycosidase enzyme breaks glycosidic bonds via acid-catalyzed hydrolysis reaction
- Beta-galactosidase hydrolyzes glycosidic bond of lactose to liberate glucose and galactose
Lock and Key theory (enzyme and substrate)
Fit together with no change in tertiary, quaternary structure
oversimplification
Enzyme and substrate “Induced fit” theory
More realistic than “lock and key”, enzyme and substrate induce conformational shifts, closer binding
Orthosteric regulation
Interacts with enzyme at active site
Lyases
cleave bonds not using hydrolysis (ex. glycolysis)
Ligases
Catalyzes formation of bonds
Feed Forward Regulation
Upstream product in higher concentration catalyzes downstream reaction
ex. In glycolysis, pyruvate kinase catalyzes ATP producing rxn, enzyme activated by intermediate fructase 1,6-bisphosphate upstream
How to lower Ea
Lowering Ea done by deploying catalysts (enzymes)
- stabilize transition state
- weaken bonds within reactants
- changing orientation to elicit effective collisions
- increasing frequency of collisions
- donating electron density to reactants
Enzymes don’t affect thermodynamics: Gibbs free energy, enthalpy, entropy
Catalysts not consumed, small amount can greatly affect products
Heterogenous vs. Homogenous Catalysis
Based on the phase of catalyst compared to phase of reactant species
-
heterogenous catalyst: in different phase than reactants
- typically catalyst is solid and reactants are liquid/gas
based on surface area of catalyst, grinding it into a powder more efficient
- homogenous catalyst: same phases
Kcat
turnover number is defined as the maximum number of chemical conversions of substrate molecules per second that a single active site will execute for a given enzyme concentration
High kcat is efficient
Sn1
Two steps:
- Leaving group leaves, generates carbocation (slow)
* carbocation formation = rate limiting step
depends only on concentration of substrate, reaction rxn rate only depends on electrophile
- Stronger carbocation = better substitution, tertiary over primary and secondary
2. Nucleophile attacks - often molecule of water or alcohol, will become positive
- next step is to deprotonate
Not stereo sensitive, can attack from front or back –> equal conc of S and R stereoisomers
- Racemic mixture of products
Rate = k[A]
Sn2
Nucleophile and electrophile involved in rate-limiting step; rxn rate depends on both their concentrations
- No carbocation is formed, weaker electrophiles
- most common substrate is alkyl halide (tert, sec, or primary)
Carbon attached to halogen has partial positive charge
- Nucleophile must attack opposite (back-side) of leaving group (halogen)
- both nucleophile and leaving group are electron rich
substrate chirality is inverted (ex. S–> R)
Rate = k[A][B]
Nucleophilic addition
Begin with nucleophilic attack, but no leaving group
- Oxygen protonated
- nucleophilic attack
* in base catalyzed, nucleophile made stronger by deprotonation - deprotonation
Common examples of nucleophiles: water, alcohols, hydrogen cyanide
- all have a spare electron pair to donate
Henderson hasselback equation- used to determine pH of buffer system
pH = pKa + log([A-]/[HA])
Anomers
If an anomer is an aldose, which carbon differs
Ketose?
C1 (where aldehyde is)
C2 (where ketone is)
