Biochemistry Ch. 10 Regulatory Strategies
zymogen activation
Blood clotting cascades are controlled by
lactate dehydrogenase or LDH
Blood serum analysis of ____________________________ isozymes is used in the diagnosis of a myocardial infarction.
How is the clotting cascade initiated?
Both intrinsic (damaged surface) and extrinsic (trauma) pathways can induce the cascade. The initial steps differ, but lead to a final, common path to form the fibrin clot.
Phosphorylation is
Catalyzed by Protein Kinase Enzyme that transfers a phosphate from ATP to R group hydroxyl on S,T,Y of protein. Serine / threonin protein kinase Tyrosine protein kinase
a bi-substrate analog that resembles the catalytic transition-state intermediate
Changes in ATCase conformation were detected by crystallizing the enzyme in the presence of PALA (N-(phosphonacetyl)-L-aspartate). What is PALA?
Carboxypeptidase
Cleaves C terminal amino acids from proteins / peptides
Elastase
Cleaves peptides on C terminus of small side chains (A,S)
Chymotrypsin
Cleaves proteins on C terminus side following aromatic (F,Y,W) or large hydrophobic (M) amino acids
Warfarin
Clinical Anticoagulant Rat poison Vit. K antagonists
Break Down of Clots
Clots dissolve when integrity of damaged area is restored. Plasmin TPA
Aspartate Transcarbamoylase (ATCase)
Committed step in multistep pathway to synthesize pyrimidines Multisubunit enzyme 2 c3 + 3 r2 → c6r6
Two Models explain Cooperativity
Concerted model Sequential model Aspartate Transcarbamoylase exhibits concerted model, but other cooperative enzymes are explained to varying extent by the two models.
Why is covalent modification advantageous when compared to proteolytic activation?
Covalent modification is usually a reversible process
Processes using Zymogens
Digestive enzymes Blood clotting Protein Hormones Collagen Development Apoptosis
Why are there Isozymes?
Encoded by different genes-Distinct proteins Expression may differ by: Organelle Tissue Developmental stage This sllows fine tuning of metabolism to meet the needs of different tissue or developmental stage. Examples Aldehyde dehydrogenase Lactate dehydrogenaase
Protein Phosphatase
Enzyme that removes a phosphate from phosphorylated protein. Examples: Serine / threonin protein phosphatase Tyrosine protein phosphatase
zymogens
Enzymes activated by proteolytic cleavage.
Isozymes (isoenzymes)
Enzymes that differ in amino acid sequence yet catalyze the same reaction. Exhibit different kinetic parameters (KM, Vmax, etc)
Give several examples of enzymes and proteins that are activated by proteolytic activation
Examples include digestive enzymes (trypsin), hormones (insulin), clotting enzymes (fibrinogen), developmental process proteins (collagen), and apoptosis proteins (caspases)
Fibrinogen (I) → Fibrin (Ia) (Cont')
Fibrin monomer assembles into fibrous arrays called protofibril. α chains contain H3N+-Gly-Pro-Arg which are bound by γ domain Protofibils interact with each other to form soft clot. β chains contain H3N+-Gly-His-Arg which are bound by β domain
Protein Kinase A (PKA) Structure
Heterotetramer of two subunits (R2C2) C catalytic subunit (38 kd) Phosphorylates target proteins (protein kinase activity) when freed by R subunit. R regulatory subunit (49 kd) Each R subunit contains two binding sites for cAMP cAMP stimulates PKA. Numerous isozymes
lysine
Histones are acetylated at specific ________________ residues.
PKA Participates in "flight or fight" response
Hormone epinephrine (adrenaline) binds membrane receptor Stimulates synthesis of cAMP (second messenger) by adenylate cyclase. cAMP concentration approaches 10nM Two cAMP bind to regulatory subunit of PKA Catalytic subunit is activated and functions as kinase
cleavage between an Arg and Ile by trypsin
How is chymotrypsinogen activated?
by binding an inhibitor protein
How is trypsin activity turned off?
Proteolysis of peptide bond between amino acid 15 and 16 cause Chymotrypsin Conformational Change
Ile16 turns inward and forms ionic bond to Asp194 Met192 moves to surface Residues 187 and 193 become extended Begins creation of hydrophobic substrate binding cleft Oxyanion hole is incomplete Hydrolysis of a single peptide bond causes discrete conformational changes.
What is the final step in the clotting pathway?
In the final step, fibrinogen, which contains six chains of three subunit types, is altered. Thrombin cleaves four of the chains, resulting in the formation of fibrin monomers. These monomers spontaneously assemble into the fibrin array. The clot is stabilized by cross-links between the amino acids catalyzed by transglutaminase.
Trypsin Inhibitor
6 kd protein Inhibits trypsin in pancreas and pancreatic duct Binds tightly to trypsin's active site An effective substrate analog (lys 15 - ala 16). Lys 15 of trypsin inhibitor interacts with asp 189 of trypsin Slow reaction rate Half life = several months
TPA
72 kd protein structure related to prothrombin (see below) Used clinically to break up clots during MI.
Trypsin Inhibitor survives common denaturing agents
8 M urea 6 M guanidine hydrochloride
Z antitrypsin
= lys 53 → gln mutant Homozygous Z individuals have 15% of alpha 1 antitrypsin as individuals with normal form of alpha 1 antitrypsin Increase activity of elastase leads to emphysema Heterozygous Z individuals that are smokers have increase risk of emphysema due to oxidation of Met 358 of alpha 1 antitrypsin
zymogen
A ______________ is an inactive precursor of an enzyme that is activated by a proteolytic cleavage.
Phosphorylation is an extremely effective tool for catalytic control. Explain the reasons.
A phosphoryl group adds negative charges, allowing new electrostatic interactions and new hydrogen-bond formation. The free energy charge of phosphorylation is large, which can affect the conformational equilibrium of different states. Using ATP means that the reaction is linked to the energy status of the cell. Phosphorylation is rapid and reversible and can result in amplified effects. These factors affect structural, thermodynamic, regulatory, and kinetic properties.
proteolytic cleavage
A regulatory mechanism that is NOT readily reversible:
heterotropic
A type of regulator of an allosteric enzyme that is not a substrate
Trypsin
Activates more trypsin and other zymogens. Hydrolyzes proteins on C terminus side following long, positively charged R groups (e.g. R, K)
Enteropeptidase
Activates trypsin by hydrolyzing unique lys 6-ile 7 peptide in trypsinogen.
How are Enzymes are regulated?
Allosteric Control Multiple forms of Enzyme Proteolytic activation Control the amount of enzyme Reversible Covalent Modification
ATCase Activity In vivo
As [substrate] increases more enzymes bind to substrate and are transformed into R state, increasing the ability to bind even more substrate
pyrimidines
Aspartyl transcarbamylase catalyzes the first step in the synthesis of ______________.
Phosphorylation / Dephosphorylation
At physiological conditions Irreversible Rate of uncatalyzed reaction is negligible Kinases / Phosphatases may be: Dedicated kinases / phosphatases Multifunctional kinases / phosphotases
Acetylation
Attachment of an acetate group to the R group of lysine Histones are acetylated and deacetylated
ATP as allosteric effector Activator (ATCase)
Binding of ATP stimulates activity. ↑ [ATP] signals ↑ [purine] ↑ energy charge available for Replication and Transcription Need for deoxy and ribonucleotides ATP competes with CTP for allosteric site
CTP as an allosteric effector Inhibitor (ATCase)
Binding of CTP inhibits enzyme activity. Substrate binding more difficult. Shifts curve to right. With CTP bound, enzyme requires ↑[S] to reach given V.
What causes ATCase's conversion from the R to T state?
Binding of CTP shifts enzyme toward the T state, thus inhibiting enzyme activity. CTP binds to domain of R subunit that does not interact with C subunit.
What causes ATCase's Conversion from T to R state?
Binding of Substrate (carbamoyl phosphate and asparatate OR PALA) C subunits move 12 Å apart Rotate 10o R subunits Rotate 15o
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 of one substrate molecule leads to transition to R state of neighboring active sites (but not ALL active sites).
Protein Kinase A Catalytic Inhibition
Inhibitor and ATP-Mg2+ bind to active site of PKA catalytic subunit. Two lobes Smaller lobe binds ATP-Mg2+ Large lobe binds protein and contains catalytic residues Binding of Substrate causes conformational change (two lobes mover closer together) in PKA.
Tissue factor pathway inhibitor (TFPI)
Inhibits TF-VIIa-Xa complex
Antithrombin III
Inhibits thrombin by forming irreversible complex. Blocks other serine proteases XIIa, XIa, IXa, Xa Stimulated by heparin Negatively charged polysaccharid3
Proteolytic activation
Irreversibly converts an inactive enzyme (zymogene or proenzyme) into active enzyme. Examples digestive enzymes and blood clotting.
Why are certain forms of lactate dehydrogenase used as heart attack indicators?
LDH is a tetramer, made of M and H isozyme subunits. The M tetramer functions in an anaerobic environment and is primarily found in skeletal tissue, whereas the H tetramer, found primarily in the heart, functions optimally in an aerobic environment. An excess of the H type in serum indicates that a heart attack occurred.
catalytic and regulatory
Many allosteric enzymes have two types of subunits, termed
γ-carboxyglutamate
Modified amino acid found in prothrombin, necessary for activation
can modify several different targets
Multifunctional protein kinases
isozymes
Multiple forms of homologous enzymes found within an organism
Do allosteric enzymes follow traditional Michaelis-Menten kinetics? Compare a graph of rate relative to substrate concentration for ATCase and compare it to Michaelis-Menten enzyme.
No, ATCase displays different kinetics. A plot of rate versus substrate concentration is a sigmoidal curve, as opposed to the simple hyperbolic curve obtained by enzymes displaying Michaelis-Menten kinetics.
Active site is Between Subunits of Catalytic Trimer
One subunit- The 53, The 55, His 134, Arg 167, Arg 229, Gln 231 Adjacent subunit- Ser 80, Lys 84
Cyclic AMP (cAMP)
Phosphate connects 3'carbon to 5' carbon. Second Messenger Example: Amplification of fight or flight signal from hormone epinephrine (adrenalin)
Phosphorylation works well to regulate activity because
Phosphate's two negative charges disrupt electrostatic interaction Phosphate bonds are directional: > 3 H bonds Tetrahedral geometry make H-bonds directional Large Free energy change. Rapid rate: < s Amplified effect Use of ATP ties phosphorylation to energy charge of cell
Multifunctional kinases / phosphotases
Phosphorylates / dephosphorylates numerous targets
Dedicated kinases / phosphatases
Phosphylates / dephosphorylates specific target
c-AMP
Protein kinase A is activated by binding ________________ to specific sites on the regulatory subunit.
tyrosine
Protein kinases add phosphoryl groups to serine, threonine, and _________________ residues in target proteins.
Prothrombin (II) → Thrombin (IIa)
Prothrombin four domains Gla domain = γ-carboxyglutamate rich domain which binds Ca 2+ Modified amino acid with two carboxylic acids Two Kringle domain = resemble pastry Serine protease domain Xa activates prothrombin by making two proteolytic cleavages
(Protein Kinase A) PKA Structure
Pseudosubstrate (Arg-arg-gly-ala-Ile) binds to the active site of the C subunit. Pseudosubstrate sequence similar sequence to the kinase recognition sequence.
PseudoSubstate binds to active site of catalytic subunit of PKA
Pseudosubstrate (yellow) Arg-Arg-Asn-Ala-Ile Arg 1 (pseudosubstrate) forms ionic bond with the carboxylate of Glu 127 (PKA) Arg 2 (pseudosubstrate) forms ionic bond with tow carboxylate groups of PKA Ile 5 (pseudosubstatrate) forms hydrophobic bonds with Leu 198 and Leu 205
Dephosphorylation
Removal of phosphate from phosphorylated protein Catalyzed by Protein Phosphatase
protein phosphatases
Removal of protein phosphates is catalyzed by ____________.
Phosphorylation
Reversible attachment of phosphate group to hydroxyl of R group of S, T, Y. Phosphate usually from gamma phosphate of ATP Turns things on or off
Reversible Covalent Modification
Reversible attachment of phosphoryl (or other group) to R group of amino acid on enzyme
Hemophilia A
Sex linked recessive Defective Factor VIII- Intrinsic pathway missing / reduced Current treatment for Hemophilia A involve transfusion with factor VIII, produced using recombinant DNA technology.
Normally Factor VIII
Simulates Factor IXa (serine protease) to activate factor X (a protease)
Fibrinogen (I) → Fibrin (Ia)
Six subunits two each (Aα, Bβ, γ) 340 kd Thrombin hydrolyzes four arg-gly peptide bonds to activate fibrinogen to fibrin monomer (αβγ)2 Four fibrinopeptides removed Two from A chain (18 residues) Two from B peptide (20 residues)
Homotrophic effect
Small changes in substrate concentration lead to large changes in activity (conversion of T state to R state)
Fibrinogen (I) → Fibrin (Ia) (Continued)
Soft clot is stabilized by formation of amide bonds between side chains of lys and gln in different monomers. Catalyzed by transglutaminase (factor XIIIa)
R subunit without Zinc separate from C subunit on sucrose gradients
Sucrose gradient centrifugation separates subunits due to differences in size. c6r6 = 11.6 S r2 = 2.8 S c3= 5.8 S
Control the amount of enzyme
Synthesize or destroy enzyme
Synthesis and Activation of Chymotrypsin
Synthesized in acinar cells of pancreas Stored in membrane bound vesicles (zymogen granules) until needed. Released into duct leading to duodenum
Aspartate Transcarbamoylase Exists in two states
T state low affinity for substrate CTP inhibits transition to R state R state Higher affinity for substrate Cooperativity At Equilibrium without substrate or CTP 200 T state : 1 R state
trypsin
The common activator of the pancreatic zymogens is
How does the sequential model differ from the concerted model for allosteric enzymes?
The concerted model does not allow for anything other than an "all-or-none" complete tense- or relaxed-form protein. In contrast, the sequential model allows for a mixed type of protein, containing some tense and some relaxed subunits. The form is in response to the ligand binding by a particular subunit
Individuals in a royal family suffered from hemophilia and often died early from bleeding. What is the cause of this disease? How is it treated?
The disease in the royal family was caused by faulty factor VIII of the intrinsic pathway. It is treated by the addition of supplemental protein, originally isolated from serum, but now a recombinant product.
homotropic
The effects of substrates on allosteric enzymes are referred to as ___________________ effects.
What is the function of aspartate transcarbamoylase?
The enzyme catalyzes the first step in the synthesis of pyrimidines. It condenses carbamoyl phosphate and aspartate to form N-carbamoylaspartate and inorganic phosphate.
protein kinases
The enzymes that catalyze protein phosphorylation
T-state
The less active conformational form of an allosteric enzyme
feedback inhibition
The name for the regulatory mechanism in which the final product inhibits activity of the first step.
How is the kinase cascade activated?
The process is often initiated by hormones that bind to membrane receptors. This process activates adenylate cylase, which causes the formation of cAMP, an intracellular messenger. The cAMP activates a key protein kinase. In eukaryotic cells this is an allosteric enzyme, protein kinase A, which then phosphorylates various target proteins
homotropic
The regulatory effects of substrates on allosteric enzymes are referred to as _______________ effects.
higher
The relaxed form of an allosteric enzyme has _________ affinity for the substrates.
sigmoidal
The shape of the kinetic plot of an enzyme that exhibits cooperative binding
The sigmodial curve of aspartate trascarbamoylase explained
The sigmodial curve exhibited by ATCase is a combination of Michaelis Menten curves for the T state (high KM for substrate) and R state (low KM for substate).
Why was it surprising to find that CTP inhibits ATCase?
The substrates for ATCase are carbamoyl phosphate and aspartate. These molecules do not resemble CTP. Thus, it was clear that the CTP must not bind to the active site, but to a distinct regulatory site.
What is the dual action of thrombin?
Thrombin catalyzes the hydrolysis of fibrinogen to form active fibrin. But it also has a role in shutting down the cascade by regulating protein C, a protease that digests other clotting enzymes Va and VIIIa.
What protein is administered after heart attacks to increase survival odds, and what is the biochemistry involved?
Tissue-type plasminogen activator, or TPA. TPA leads to the dissolution of blood clots so that blood flow through the blocked coronary artery can be restored. TPA activates plasminogen that is bound to the fibrin clot to active plasmin, which then hydrolyzes the fibrin of the clot.
Cascade Activating Blood Clotting Extrinsic pathway
Trauma exposes tissue factor (TF) an integral membrane glycoprotien.
Cascade Activating Blood Clotting Intrinsic pathway
Triggered by exposure of anionic surfaces on rupture of endothelial lining of blood vessels
Lactate Dehydrogenase
Two forms H form (heart) M form (muscle) 75% identical-Differential expression through development Functions as Tetramer Combinations of subunits H4- higher KM and allosterically inhibited by pyruvate M4- Lower KM and not inhibited by pyruvate Intermediate KM, inhibition by pyruvate
Ethanol Sensitivity
Two isozymes of Aldehyde dehydrogenase Mt form-Low KM Cytoplasmic form-High KM Sensitive people have a less active mitochondrial enzyme due to an amino acid substitution; Therefore, acetaldehyde is processed only by the cytoplasmic enzyme. With high KM this enzyme achieves a high rate of catalysis only at very high [acetaldehyde]. Acetaldehyde in blood causes facial flushing and tachycardia (rapid heart beat)
Common Covalent Modifications
Used to turn proteins "on or off"
Dicoumarol
Vit. K antagonists Compound found in spoiled sweet clover. Fatal hemorrhagic disease in cattle. Cattle synthesize abnormal prothrombin that does not bind Ca2+
vitamin K
Vitamin required for the clotting pathway
serine tyrosine threonine
Which amino acid residue side chains is/are phosphorylated by protein kinases?
pepsinogen procarboxypeptidse
Which of the following is an example of a zymogen?
ATP
____________ is the most common donor molecule in phosphorylation reactions catalyzed by protein kinases.
Isozymes or Isoenzymes
_______________ are multiple forms of homologous enzymes within the same organism that catalyze the same reaction but with different kinetic properties.
Plasmin
activated by TPA (tissue-type plasminogen activator) cleaving plasminogen Serine protease that hydrolyzes peptide bonds in fibrin clots
Alpha 1 Antitrypsin
alpha 1 antiproteinase antielastase. 53 kd protein synthesized in liver function in plasma Inhibits elastase (and other proteases) Normal Alpha 1 antitrypsin with methionine sulfoxide does not inhibit (bind) elastase
ATCase's 2 catalytic (c3) subunits
c3 = trimer (34 kd each) 2 C subunits stacked on top of one another Each C subunit contains 3 active sites (6 active sites / enzyme molecule) Purified C subunit exhibits catalytic activity but unresponsive to CTP. Exhibits Michaelis Menten Kinetics (hyperbolic curve) by itself
Allosteric proteins
contain distinct regulatory sites and have multiple functional sites. display cooperativity
Lipase
digests lipids
Characteristics of Allosteric Enzymes shown in Aspartate Transcarbamoylase
do NOT exhibit Michaelis Menten Kinetics Plot of V vs [S] for allosteric enzyme produces: Sigmoidal curve exhibits concerted model Activity affected by binding of affecter at allosteric site
Allosteric Control
exhibit cooperativity - activity at one function site affects the activity at other sites Example Transcarbamolyase (ATCase)
Increase in ratio of H4 to H3M
in blood is used to detect MI (myocardial infarction)
Aspartate Transcarbamoylase and CTP
inhibited by CTP ↑ [CTP] = ↓ activity (Velocity or rate) CTP does NOT bind to active site binds to allosteric (regulatory) site Classic example of Feed back inhibition
Multiple forms of Enzyme
isozymes (isoenzymes) each form may have different KM and Vmax values. Example lactate dehydrogenase
The most common strategy(ies) for enzymatic regulation:
multiple enzyme forms proteolytic activation allosteric control reversible covalent modification
Zymogens
or Proenzyme inactive precursor of enzyme often activated by proteolysis
cysteine
p-Hydroxymercuribenzoate reacts with crucial ______________ residues in ATCase
Subunits without zinc separate on sucrose gradients (Cont)
p-hydroxymercuribenzoate is removed from subunits by adding mercaptoethanol catalytic and regulatory subunits are recovered. Activity restored Catalytic and regulatory subunits may be studied separately
dissociation of catalytic (C ) and regulatory (R ) subunits in ATCase
p-hydroxymercuribenzoate reacts with sulfhydryl group of cysteine residues to break bonds to zinc Breaking of bonds between zinc and cysteine allows dissociation of catalytic (C ) and regulatory (R ) subunits.
Examples of covalent modification include
phosphorylation and dephosphorylation acetylation ubiquination
ATCase's 3 regulatory (r2) subunits
r2 = dimer (17 kd each) R subunit binds 2 CTPs (allosteric inhibit); stabilizes the T form R subunit contains 2 Zn 2+ binding sites Each C subunit contacts two R subunits at a structural domain in the R subunit that is stabilized by a Zn2+ ion bound to 4 cysteine residues
Secretory Pathway
rER- Proteins (zymogens) synthesized Transport vesicles- COP II Golgi- Clathrin coated Storages vesicles (granules)- Stored until nerve impulse or hormonal signal is received Secretion
PALA N-(phosphonacetyl)-L-aspartate
resembles a reaction intermediate Competitive inhibitor of Aspartate Transcarbamoylase Binds at active site
Serpins
serine protease inhibitors family of inhibitors. Alpha-1-Antitrypsin Antithrombin III
What do cigarette smoking and elastase have in common?
α1-Antitrypsin is a neutrophil protein that inhibits elastase. Some individuals with a genetic disease causing deficiency of functional α1-antitrypsin have scarred lungs due to overactive elastase. One side effect of smoking is oxidation of a met in the α1-antitrypsin, and it no longer functions efficiently as an inhibitor. Thus, both a genetic disorder and smoking can lead to emphysema.
Prothrombin (II) → Thrombin (IIa) (Cont')
γ-carboxyglutamate binds Ca2+ The enzyme that adds the second carboxylic acid requires vitamin K K = Koagulation (Scandinavian spelling) Ca2+ causes prothrombin to attach to phospholipid membranes derived from blood platelets after injury
