502 midterm Flashcards
Catalyst
speed up rate of rxn but are unchanged in the process. ALL enzymes are protiens, except RNA catalysts (peptidyl transferase)
why we need enzymes
- most of biochem rxns in body depend on them
- In living organisms, no biochemical reaction would run without an enzyme.
- Inborn errors of metabolism are due to genetically determined abnormalities of enzymes. Thus, knowing the enzyme determines the treatment. Creatin kinase is example of this
- Diagnostic enzymology uses enzymes to assist in diagnosis.
- Enzymes can also be used in therapy. Example: enzymes used as therapy in stroke to break up blood clots
- There are endless uses of enzymes in research laboratories.
- W/o enzymes, rxns would run at a very slow rate. Enzymes increase rxn speed between 10^4-10^16 speed
Substrate
substrate is the molecule acted on by the Enzyme to generate a product. Enzyme binds the substrate at it’s active site (substrate binding site)
Active site
contains subsrate binding site and catalytic site. Substrate binding site has substrate specificity while catalytic site has catalytic residues that act on the subsrate and has reaction specificity.
Substrate binding site
has specificity for substrate based on 3-D conformation and R-group specificity in that area
Catalytic site
at this site, we could have R-groups that are donating electrons or accepting them to allow the rxn to proceed
Allosteric Site
NOT THE ACTIVE SITE, some other site on the enzyme where a effector molecule can bind and result in conformational change in enzyme that leads to increased or decreased activity
Isoenzymes
- Isoenzymes are enzymes that catalyze the same reaction but differ slightly in amino acid composition, since they arise from different genetic loci.
- Diagnostic value: Different organs may contain characteristic proportions of isoenzymes, so isoenzyme levels in blood may serve to identify the exact site of tissue damage.
- The amino acid composition, thus the net charge of these subunits are different, consequently, the net charge of the enzyme complex will also be different Electrophoretic separation of these isoenzymes is possible. In this case, we do not add SDS (no neg charge given to them) so the enzymes only move based on their own net charge at a given pH. Thus pH of buffer is adjusted to allow for good separation of these isoenzymes.
Enzyme specificity:
- The high specificity of enzymes lies in the specific side chains in the substrate binding site and catalytic sites and in the overall tertiary structure.
- Substrate binding site determines the kind of substrate that can bind to the enzyme;
- The catalytic site determines the character of the reaction that is catalyzed by the enzyme.
Creatine Kinase
- Highest concentration of it is in muscles
- CK - creatine + ATP creatine-phosphate + ADP
- CK is a dimer with two types of subunits:
- M (short for muscle)
- B (short for brain)
- The 2 subunits can be combined in 3 different ways - BB = CK 1 brain (and colon), MB (CK2 myocardium), and MM (CK3 skeletal muscle and myocardium.
- CK-MB is important for diagnosing re-infarction because in begins to normally fall after a day so subsequent elevation means that another event is happening. The 3 different types can also be separated with electrophoresis. BB form has highest neg charge so it moves furthest, MM form has less neg charge so moves least far and MB form would be somewhere in between.
Holoenzyme
apoenzyme +coenzyme
Apoenzyme
the protein part of an enzyme
Cofactor
- small inorganic molecule that participates in rxn
inorganic metal ions: - iron (Fe), zinc (Zn), cobalt (Co), calcium (Ca), magnesium (Mg), copper (Cu)
Coenzyme
- small organic molecules, derived from water-soluble vitamins
- Both cofactors and coenzymes bind to the enzyme close to/at its active site. This binding can be apolar interaction with the protein, covalent binding or coordinative binding (metal ions).
Induced fit model
- Substrate comes into the active site and causes a small conformation change in the enzyme
ways to increase rate of rxn
- lower activation energy
- increase temp
- increase amount of substrates
Serine Proteanases
- trypsin, chymotrypsin , elastase
- Difference between serine proteinase for trypsin vs chymotrypsin and elastase is that at the substrate binding site, the AA that are recognized are different, this is substrate specificity. For trypsin, polypeptide chain binds in active site and Trypsin will cleave amino acids after BASIC AA residues (so arginine, histidine, lysine)(which is why the Asp189 below is – charged. Chymotrypsin cleaves aromatic AAS. Elastases cleaves elastin.
Km
Km = 1/2 Vmax
- Km is the substrate concentration at which the enzyme works with half of its maximal velocity. So Km = ½ Vmax
- Km - physiologic significance: The lower the Km, the more efficient the enzyme is in binding the substrate.
- Km can also be described as the affinity of the enzyme to its substrate.
- Units for Km is substrate concentration
Km= {k2 + k3} / k1 Michaelis-Menten constant. Km is basically the constant for the dissociation of the ES complex. Top part of equation is the rate constants for breakdown of ES and bottom part is rate constant for formation of ES complex.
MM kinetics 3 assumptions
- the ES is always in steady state (concentration is constant, (for when substrate concentration is higher, which is usually true b/c enzyme concentrations are generally lower)
- all of the E is in the ES form (enzyme is saturated) when substrate is at a high concentration; ); (formation of ES complex = breakdown of the ES complex)
- when all enzyme is in ES complex, the rate of formation of products is maximal or Vmax=k3[ES].
Vo
initial velocity - slope of the line at the beginning of MM curve
¥ Velocity is the change in concentration of substrate or product per unit time.
¥ How much P is formed – depends on how much substrate is available
¥ How fast the P is formed – depends on how much Enzyme is available
Vo can be found based on how much product has been made, Product < 0.05 x Substrate
Km and affinity trend
Higher affinity means a lower Km and lower affinity means a higher Km value. At high affinity, you can pick up and use substrates at lower substrate concentrations
Lineweaver-Burk plot
The slope is Km/Vmax
the Y intercept is 1/Vmax
the X intercept is –1/Km
Equation: 1/v0 = Km/Vmax x 1/[S] + 1/Vmax
Effect of Temp
Effect of pH
Effect of temp: Curve is bell shaped because velocity is increased at high temp but past this best temp range the enzyme denatures so reaction stops
Effect of pH: Shown is two phosphatases , an alkaline and an acidic phosphatase, so charges on the sidechains reacting with these will be diff. These enzymes are pH depenedent on a certain pH range and don’t work in other ranges
Regulation of enzyme-catalyzed reactions
A. Availability of substrate and coenzymes
B. Amount of enzyme
C. Activity of enzyme
Regulating enzyme activity :covalent modification
C.1. covalent modification
C.1.1. irreversible – cleavage
C.1.2. reversible – ex is phosphorylation event
Regulating enzyme activity :non-covalent modification
C.2. non-covalent modification
C.2.1. inhibition
C.2.2. allosteric modification
competitive inhibition
Completive inhibition: v max is same but Km is different, Km increases for comp inhibition because there is less affinity at substrate binding site
- Competitive inhibitors are inhibitors that are structurally similar to the substrate and bind at the substrate-binding site.
Non-competitive inhibition
Non competitive inhibition – Km is same but Vmax is different, Vmax decreases for noncomp inhibition
- A noncompetitive inhibitor binds at the catalytic site.
allosteric control
- Effector binds to a place NOT ON THE ACTIVE SITE. This causes a conformational change in the enzyme
- Can be an activator site or and inhibitory site
- V class of effectors effect Vmax
- K class of effectors effect Km so they effect affinity of enzyme to the substrate
a. Hydrophobic amino acids
i. Alanine (Ala or A)
ii. Valine (Val or V)
iii. Isoleucine (Ile or I)
iv. Leucine (Leu or L)
v. Methionine (Met or M)
vi. Phenylalanine (Phe or F)
vii. Tyrosine (Tyr or Y)
viii. Tryptophan (Trp or W)
a. Hydrophilic amino acids
Basic
- Lysine (lys or K)
- Arginine (Arg or R)
- Histidine (His or H)
+ charge r group
Hydrophilic Acidic AAs
- Aspartate (Asp or D)
- Glutamate (Glu or E)
- charge r group
Hydrophilic polar uncharged
- Serine (Ser or S)
- Threonine(Thr or T)
- Asparagine (Asn or N
- Glutamine (Gln or Q)
Special AAs
Cystine (Cys)
Glycine (Gly)
Proline (Pro)
What is pka of carboxyl group
a. Pka of the carboxyl group is 2.4-2.5 so below that then that carboxyl group is protonated and there is no charge to that group
b. Above 2.4 then you lose that proton and it becomes negatively charged
What is pka of amino group
a. Above 9.6 the amino group has no charge it isn’t protonated
b. Below 9.6 the amino group pics up a hydrogen ion and becomes NH3+ so now that amino is positively charged
isoelectric point
a. 2.4
Motif
special combos of secondary structure
i. Helix-loop-helix(EF hand)-is a calcium binding motif
ii. Zinc finger- contains two antiparallel beta strands bound to a helix through a zinc ion-found in RNA and DNA binding proteins
iii. Coiled coil found in many proteins where two alpha helices are side by side
post translation protein modifications
- Acetylation – like acetylation of histones in chromosomes
- Methylation – represses transcription in chromosomes
- Carboxylation of gamma carbon in glutamate – requires vitamin K – makes gamma carboxyl glutamic acid. Lots of glu residues in osteocalcin and prothrombin (clotting
factor) . That’s why coumarin drugs inhibit vitamin K (blood thinner) - Phosphorylation – done by either kinases (requires ATP) or removed by phosphatases (requires H2O). Adds a negative charge to the protien , this changes local charge in the area and could lead to conformational change which may lead to one of the basic AAs being moved towards that phosphate group, which lead to change in activity of protein.
- Proteolytic processing - signal peptide cleavage, intracellular or extracellular polypeptide cleavage, proenzyme activation
- Protein glycosylation – carbohydrate addition to proteins is nontemplate. This requires acceptor protien, glycosyl transferase and an activated sugar
