Enzyme Mechanisms Flashcards
What is the active site of a molecule?
The place where the enzyme binds. Often a pocket or cleft surrounded by amino acid residues that help bind the substrate (and other residues that play a role in catalyst).
How does a substrate recognize the active site?
Through complimentarity.
What is the Lock and key hypothesis of enzyme binding and why did it suck?
The enzyme accommodates the specific substrate as a lock does a specific key. Increased understanding in enzyme specificity but didn’t help our understanding of catalyst because a lock does nothing to its key
What is the Induced fit hypothesis?
The enzyme doesn’t just accept the substrate it, the enzyme also demands that the substrate be distorted into something close to the transition state.
What must an efficient catalyst do in regards of the transition state?
An efficient catalyst will stabilize the transition state and thereby lower its free energy relative to other states.
What are the four basic steps in enzyme binding? [enzyme (E), substrate (S), product (P)]
E+S -> ES -> EP -> E+P
What are the three genera l things that all enzymes do?
1.) binds to substrate or substrates. 2.) Lowers the energy of the transition state. 3.) Directly promotes the catalytic event. when the catalytic processes is over the enzyme must be able to release its original product and return to its original state
Is formation of the enzyme-substrate complex thermodynamically favorable? Why?
Oh yeah, because of complementarity.
What are the 6 ways an enzyme may achieve rate enhancement?
- ) preferred binging to the transition state through complementarity noncovalent bonding interactions (H-bonds, charge-charge, etc.) (NC- bonds are electrostatic in nature so this is called electrostatic catalysis.)
- ) Distortion of the substrate and/or active site which premotes reduction of the activation energy (induced fit)
- ) binding of substrates to optimize proximity & orientation (making ΔS °# more favorable)
- )altering the reaction pathway to include intermediate states. (typical of covalent catalysis)
- ) general acid/base catalysis (GABC) important in reactions involving proton transfer.
- ) metal ion catalysis
Whats a general acid in enzyme hell?
Donates an H+ to an atom that develops a negative charge in the transition state.
Whats a general base in enzyme hell?
Removes an H+ from a atom that develops a positive charge in the transition state
Whats special about histadine in GABC?
It can donate protons at a physiological pH.
What are Metalloenzymes?
Enzyme with a metal ion at its active site.
Where is the active site of lysozyme located?
In a deep cleft
How many glycosyl residues bind to lysozyme?
6 reidues bind to substituents A-F
Where does lysozyme cleave the glycosidic bond?
In between residues D-E
How to describe an amino acid mutation of glutamic acid replacing glutamine at residue 35 in single letter code?
E35Q
What is Lysozymes optimum pH?
~5
Whats a serine protease?
Important class of enzymes. Called protease because they catalyze the hydrolysis of peptide bonds in polypeptides and proteins. This general class of protease is unique because they all have a critical serine nuleophine in the active site. Hydrolyze a large variety of esters.
What is a scissile bond in a serene protease?
A very specific binding of a particular amino acid that serves to place the active site serine very close to the carbonyl group of the bond to be cleaved.
What is a catalytic triad?
Common feature of serine protease. Has a nucleophile, general base, and acid (in many theyre composed of serine, histidine, and aspartic acid residues presented in similar 3D orientation in the active site.)
What is a Low-barrier hydrogen bond?
Shorter, 3-6 times stronger, w/ more covalent character.
What is conformational selection (selected fit)?
Implies that the unbound enzyme has multiple conformations but the substrate can only bind to an unbound enzyme with the same conformation.
What are the two general categories of “tailor made” catalyst?
Rational design and directed evolution
Example of tailoring a catalyst by James Wells.
Research focused on a specificity pocket in a protease (subtilisn). The site is usually occupied by gly. Protease comes thru and cleaves the “polypeptide chains next to the bulky hydrophobic residue”. When gly is replaced with lys this cleavage happens 500x faster.
What are fusion proteins?
Proteins coded for by genes that have been spliced together in vitro from two or more sources.
What are cofactors?
A cofactor is a non-protein chemical compound or metallic ion that is required for a protein’s biological activity to happen.
Cofactors are not irreversibly changed during catalysis; they are either unmodified or regenerated.
How do cofactors relate to organisms in general?
Cofactors often have complex organic structures that cannot be synthesized by some organisms – mammals in particular.
What is the vitamin-B complex?
The collective name for the water soluble vitamins that are metabolic precursors of several cofactors in the body.
B1, B2, B12, Niacin, and more.
What is NAD+?
NAD+ is nicotinamide adenine dinucleotide
It is derived from the vitamin niacin
What is the active part of NAD+?
The nicotinamide portion is capable of being reduced and serves as an oxidizing agent to which two eletrons and a proton are added to the nicotinamide ring
What does alcohol dehydrogenase do?
Turns ethanol to aldehyde
How does NAD+ act as a cofactor? Example
When it is reduced to NADH and then reoxidized to regenerate the NAD+ all within the same enzyme/catalytic reaction
How does NAD+ act as a substrate? Example
When it is turned into NADH and then has to go to another enzyme to be reoxidized and become NAD+ again
What are metalloenzymes?
They are enzymes that contain one or more metal ions
Usually held by coordinate covalent bonds from amino acid side chains, but sometimes bound by a prosthetic group like heme.
What are the six major classes of enzymes?
- Oxidoreductases
- Transferases
- Hydrolases
- Lyases
- Isomerases
- Ligases
What do oxidoreductases do?
- Catalyze oxidation-reduction reactions
What do transferases do?
Catalyze transfer of functional groups from one molecule to another
What do hydrolases do?
Catalyze hydrolytic cleavage
What do lyases do?
Catalyze removal of a group from or addition of a group to a double bond, or other cleavages involving electron rearrangement
What do isomerases do?
Catalyze intramolecular rearrangement
What do ligases do?
Catalyze reactions in which two molecules are joined
What are ribozymes?
RNA molecules that can act as enzymes
What is ribonuclease P and what does it do?
Ribonuclease P is an RNA-protein complex that catalyzes the hydrolysis of a phosphodiester bond which is involved in the production of tRNA from pre-tRNA.
The RNA portion can do this by itself.
Are there DNA that act as enzymes?
None have been found in nature.
Some have been developed in labs
What is substrate-level control?
A type of enzyme regulation that occurs when the products of an enzyme interact with the substrate of that enzyme itself.
Usually inhibits
What is feedback inhibition?
An increase in the concentration of E leads to a decrease in its rate of production by inhibiting the enzyme that converts A to B
A->B->C->D->E
What is allosteric regulation?
Allosteric regulation is the regulation of an enzyme by binding an effector molecule at a site other than the enzyme’s active site.
What is homoallostery?
Cooperativity in substrate binding. Binding of one substrate favors binding of additional substrates.
For example, hemoglobin
What is heteroallostery?
If an enzyme can exist in two conformational states, T and R, then:
Allosteric inhibitors shift the equilibrium toward T
Allosteric activators shift the equilibrium toward R
What is ATCase?
An enzyme called aspartate transcarbamoylase
What is covalent modification in the context of enzymes?
- Some enzymes are inactive until they are covalently modified
- Some enzymes are inactivated by the covalent modification
- Some covalent modifications can be reversed, others not.
What are protein kinases?
Protein kinases are ATP-dependent enzymes that add a phosphoryl group to the OH group of a Tyr, Ser, or Thr on some target protein
What are phosphatases?
They reverse the process of protein kinases: phosphatases hydrolyze side chain phosphate esters, releasing Pi
What is acetylation?
The transfer of an acetyl group from acetyl-coenzyme A
What is a zymogen?
- Catalytically inactive molecules that must be cleaved in order to yield active enzymes
How does stereospecificity come into play with enzymes?
The asymmetric surface of an enzyme can confer stereospecificity in the reaction of a symmetric substrate.
If the substrate molecule X2 makes at least make three contacts with unique complementary groups on the enzyme, its two X atoms are no longer equivalent.
Only a specific one of the two X atoms can contact the surface properly.
What is the proposed mechanism for UDP-galactose epimerase?
- UDP-galactose is bound to the enzyme, which carries the coenzyme NAD+.
- Hydride is transferred to NAD+ from C4 of the galactose ring to produce the carbonyl intermediate.
- Hydride is then transferred back to C4 to give the opposite stereochemistry.
- The product, UDP-glucose, is released
How do ATP and CTP regulate ATCase?
- ATP is an activator.
- CTP is an inhibitor.
Activation of chymotrypsinogen:
A series of cleavages produces the enzyme chymotrypsin, with the disulfide bonds continuing to hold the structure together.
The initial cleavage between amino acids 15 and 16 (arrow) results in the formation of p-chymotrypsin.
Subsequent removal of the segments shown in black yields a-chymotrypsin.
What sort of catalytic activation does blood clotting use?
Blood clotting involves a cascade of proteolytic activation of specific proteases, culminating in the transition of fibrinogen to fibrin.
Where does the proteolytic activation to blood clotting start?
Intrinsic pathway: The cascade of proteolytic activations starts from exposure of blood at damaged tissue surfaces.
Extrinsic pathway: The cascade of proteolytic activations starts from internal trauma to blood vessels.
