Chpt 10 Flashcards
<p>Aspartate Transcarbamoylase</p>
<p>1) Allosteric Regulation- Commited step in multistep pathway to synthesize pyrimidines
2) Multisubunit Enzyme 2C3 + 3r2-> C6R6
2 catalytic subunits
-c3=trimer
a)2 C subunits stacked on top of one another
b)each subunit contains 3 active sites (6 active sites/enzyme molecule)
-Purified C subunit exhibits catalytic activity but unresponsive to CTP
a) exhibits Michaelis menten Kinetics (hyperbolic curve)
3 regulatory subunits
-R2=dimer
a)R subunit binds 2 CTPs (allosteric inhibit); stabilizes the T form
b)R subunits contain 2 Zn2+ binding sites
Each C subunit contacts 2 R subunits at a structural domain in the R subunit that is stabilized by a Zn2+ ion bound to 4 cysteine residues
3) Active Site-is between subunits of catalytic trimers
- one->2 Thr and Arg, 1 His Gln
- adjacent-> Lys and serine
4) Exists in two States: Alternate between T and R state
-T state- Low affinity (High Km) for substrate; CTP inhibits transition to R state, thus stabilizes T state
-R State- high affinity (Low Km) for substrate; (cooperativity)
@ equilibrium w/no CTP or Substrate 200 T State: 1 R
5) Binding of Substrate causes a conformational change/conversion from T state to R state
-substrate= carbamoyl Aspartate/Phospohate or PALA
-c subunit moves 12A apart and rotate 10 degrees
-r subunit rotates 15 degrees
PALA
bisubstrate analogue
-resembles a reaction intermediate
-competitive inhibitor of Aspartate Transcarbamoylase binds at active site
6) Binding of CTP to domain of R subunit that doesn't contact c subunit shifts enzyme toward T state (stabilizing), thus inhibiting enzyme activity
7) exhibits concerted model of cooperativity, but other cooperatively enzymes are explained through varying extent by the two models</p>
Sucrose gradient Centrifugation of ATCase
Sucrose Gradient Sentrifugation-sepeart by size difference
1) Use p-hydroxymercuribenzoate
- Reacts with sulfzydrl group of cysteine residues to break bonds to zinc, which allows dissociation of catalytic (C) and Regulatory (R) subunits
2) P-hydroxymercuribenzoate is removed from subunits by adding mercaptoethanol
- catalytic and regulatory subunits are recovered and activity is restored and the two subunits can be studied seperately
R subunit without Zinc separate from C subunit on sucrose gradients which separates subunits due difference in size
Characteristics of Allosteric Enzymes
-Activity affected by binding of affector at allosteric site
Aspartate Transcarbamoylase is inhibited by CTP
- increase in CTP conc decreases activity (velocity or rate)
- CTP does not bind to active site; binds to allosteric (regulatory) site
- Classic example of Feedback inhibition
- DOES NOT exhibit Michaelis Menten kinetics; instead has a sigmoidal curve
Two models explain cooperativity
<p>Concerted Model
-Binding of one substrate molecule leads to "all or none" transition to R state. All active sites transition to R state
Sequential Model
-binding site of one substrate molecule leads to transition to R state of neighboring active sites ( but not all active sites)</p>
Sigmoidal Curve Explained
The sigmoidal curve exhibited by aspartate Transcarbmoylase is a combination of Michaelis Menten curves for the T state (High Km for substrate) and R state (Low Km for substrate)
-Homotrophic effect-small changes in substrate conc lead to large changes in activity (conversion of T state to R state)
CTP and ATP as Allosteric effector
CTP-Allosteric effector inhibitor
- binding of CTP inhibits the enzyme activity
- Substrate binding more difficult-> Right Shift
- with CTP bound, enzymes require increased substrate conc to reach given velocity (Vmax?)
ATP
Allosteric Effector Activator
-Binding of ATP stimulates activity-Left Shift
-Increase in ATP conc signals an increase in purine conc, which increases energy charge available for replication and transcription (needed for deoxy and ribonucleotides)
ATP completes with CTP for allosteric site
Multiforms of enzymes
1) isozymes(isoenzymes)
- enzymes that differ in amino acid sequence yet catalyze the same reaction.
- exhibit different kinetic parameters (Km, Vmax, etc)
2) Encoded by Different genes-Distinct proteins
3) Expression may differ by:
- organelle
- tissue
- developmental stage
4)Allows fine tuning of metabolism to meet the needs of different tissue or developmental stage
Ex: Aldehyde Dehydrogenase and Lactate Dehydrogenase
<p>Ethanol Sensitivity </p>
use alcohol dehydrogenase and aldehyde dehydrogenase
Two forms of aldehyde dehydrogenase
- Mitochondrial Form->low Km
- Cytoplasmic Form-> High Km
Sensitive people have less active mitochondrial enzyme due to an amino acid substitution, therefore acetaldehyde is only processed by the cytoplasmic enzyme.
-with high Km, this enzyme achieves a high rate of catalysis only at very high conc of acetaldehyde
Acetaldehyde in blood causes facial flushing and tachycardia (rapid heart beat)
<p>Lactate Dehydrogenase</p>
Two forms:
- H form (heart)
- M form (muscles); 75% identical; differential expression through cdevelopment
Functions as a tetramer: combinations of subunits
1) H4
- higher Km
- allosterically inhibited by pyruvate
2) M4
- lower Km
- not inhibited by pyruvate
3) Intermediate Form
- intermediate Km
- inhibited by pyruvate
<p>Detecting Damage from Lactate Dehydrogenase</p>
Presence of tissue specific isozyme in blood may signal tissue damage
-increase in ration of H4 to H3M used to detect MI (myocardial Infarctions)
<p>Phosphorylation and Dephosphorylation </p>
PHOSPHORYLATION
- reversible attachment of phosphate group to hydroxyl of R groups of S, T, Y
- Phosphate usually from gamma phosphate of ATP
Catalyzed by Protein Kinase
- enzyme that transfers a phosphate from ATP to R group of hydroxyl on S, T, Y of protein.
a) Serine/threonine protein kinase
b) Tyrosine protein kinase
Phosphorylation works well to regulate activity because:
- phosphates two negative charge disrupt electrostatic interaction
- phosphate bonds are directional
- Large free energy change
- Rapid rate-less than seconds
- Amplified effect
- Use of ATP ties phosphorylation to energy charge of cell
DEPHOSPHORYLATION
-Removal of phosphate from phosphorylated protein
Catalyzed by Protein Phosphatase
-enzyme that removes phosphate from phosphorylated protein
a)Serine/Threonine protein phosphatase
b) Tyrosine protein phosphatase
PHOSPHORYLATION/DEPHOSPHORYLATION
At physiological conditions:
-irreversible
-rate of uncatalyzed reaction is negligible
Kinases/Phosphatases may be:
1) dedicated kinases/phosphatases
- Phophorylates/Dephosphorylates specific target
2) Multifunctional kinases/phosphatases
- Phosphorylates/dephosphorylates numerous targets
<p>Acetylation</p>
- Attachment of an acetate group to the R group of Lysine
- Histones are acetylated and deaceltylated
<p>Cyclic AMP (cAMP)</p>
- phosphate connects 3’ carbon to 5’ carbon
- second messenger
- Example: amplification of fight or flight signal from hormone epinephrin (adrenaline)
<p>Protein Kinase A (PKA)</p>
<p>1) Heterotetramer of two subunits (R2C2)
C catalytic subunit
-phosphorylates target proteins (protein kinase activity) when freed by R subunit
-inhibitor and ATP-Mg2+ bind to active site of PKA catalytic subunit
-Contains 2 lobes:
a) Smaller lobe binds ATP-Mg2+
b)larger lobe binds protein and contains catalytic residues
-R regulatory subunit
a) each R subunit contains two binding sites for cAMP
Binding of substrate causes conformational change in PKA that results in the two lobes move closer together
2) cAMP situates PKA
3) Numerous Isozymes
4) PKA participates in "fight or flight" response
- hormone epinephrine (adrenaline) binds membrane receptor
- stimlutes synthesis of cAMP (second messenger) by adenylate cyclase
- Two cAMP bind to regulatory subunit of PKA
- Catalytic subunit is activated and functions as kinase
5) Pseudosubstrate binds to active site of C subunit
- pseudosubtrate seq similar to the sequence of kinase recognition seq</p>
<p>Zymogens</p>
Zymogen or Proenzyme
- inactive precursor of enzyme
- often activated by proteolysis
Processes using Zymogens
- Digestive enzymes
- Blood clotting
- Protein Hormones (EX:preproinsulin-proinsulin-insulin)
- Collagen
- Development
- Apoptosis
All enzymes secreted as zymogens use Secretory Pathway
- rER->proteins (zymogens) synthesized
- Transport vesicles (COP II)
- golgi-calthrin coated
- Storage vesicles (granules)-stored until nerve impulse or hormonal signal is received
- Secretion
