Biochem exam (ch 6-7)

Metabolic enzymes

Catalyze biochemical reactions involved in energy.

• Lowers activation energy

• Increase rate of product formation (Kinetics are effected not thermodynamics)

• Does not alter the equilibrium concentration of products and reactants

Structural proteins

Most abundant proteins

• Maintains integrity of the cell structure and promotes changes in cell shape

• Serves as the framework for individual cells, tissues, and organs

Cytoskeletal proteins

Structural proteins that are responsible or cell shape, cell migration and cell signaling

Ex. Actin, Tubulin, Collagen

Actin

Abundant cytoskeletal protein, found in muscle, subunits self-assemble from actin monomers and form long polymers called thin filaments

Can be observed under a 10x microscope and are quite large

Tubulin

An abundant cytoskeletal protein in animal cells

Self-assemble from Tubulin monomers and form long polymers called microtubules

Microtubules act as roads for movement of organelles and chromosomes during cell division

Collagen

A structural proteing (the major structural protein in animals)

Primary component of connective tissue

Gives strength to tendons, cartilage, bones, and teeth

Transport proteins

Facilitates movement of molecules within and between cells

Abundant in the plasma membrane (permit polar and charged molecules to enter and exit the cell)

Have two basic classes; Passive and active

Passive transporter proteins

Energy independent

Allow molecules to move across a membrane in response to chemical gradients

Ex. Porins or ion channels

Active transport proteins

Require energy to induce a conformational change in the protein that opens or closes a gated channel

Pump small molecules or ions against a concentration gradient

Energy comes from either ATP hydrolysis or ionic gradient

Cell signaling proteins

Transsmit extracellular and intracellular signals functioning as molecular switches

Include:

• Membrane receptors (G protein-coupled receptors GPPCR)

• Receptor tyrosine kinases

• Nuclear receptors

• Intracellular signaling

Membrane receptors

G protein coupled receptors

Receptor tyrosine kinases

Growth hormone receptors

G protein-coupled receptors

Include adrenergic receptors, such as those that bind to epinephrine-related ligands

Receptor tyrosine kinases

Play a role in the insulin pathway

Growth hormone receptors

Form dimers in the membrane (cell surface) upon binding to the growth hormone polypeptide

Erythropoietin

Induces the production of RBC

Originate in bone marrow

RBC filtered through spleen

Why is Erythropoietin and blood doping an advantage

This increases the oxygen concentration in your blood, allowing your muscles to perform better. Does have risks of DVT or PE though

Nuclear receptors

Transcription factors that regulate gene expression in response to ligand binding

Include steroid receptors such as estrogen or progesterone

Intracellular signaling protein

Functions as molecular switch, which undergoes conformational changes in response to incoming signals, such as receptor activation

Ex. Include Adenylate cyclase and protein kinases

Protein kinases (general description)

Reversibly phosphorylate proteins at Ser and Thr amino acid residues on downstream target proteins in response to upstream receptor activation signals

Phosphytase takes off the phosphate that kinase bound (Kinase is not irreversible by the same molecule but the phosphate group can be taken off)

Include

• Mitogen-activated protein (MAP) kinases

• Protein kinases A (PKA)

• Src kinases

• Phosphoinositide-3 kinase

Genomic caretaker proteins

Maintain the integrity and accessibility of genomic information

Important in repairing mutations in DNA and reproductive cells, which will be inherited by offspring

Includes proteins involved in DNA replication, repair, and recombination

• DNA polymerase - involved in replication

• DNA ligase - binds the transcription strand to where the primer was

• Topoisomerase

• DNA primase - start replication process

• Photolyase

Involved in gene expression

• RNA polymerase

RecBCD

A genomic caretaker protein that allows our DNA to be accessable for replication

Myoglobin

Concentrated in muscle

It is a storage depot for O2 but its not accessable

Binds oxygen reversibly to Fe2+ in a porphyrin ring tightly bound to the protein

Hemoglobin

Major protein in RBC

Makes up 35% of dry weight of red blood cells

Transports heme bound O2 from lungs and tissues through the circulatory system

Binds oxygen reversibly to Fe2+ in a porphyrin ring tightly bound to the protein

Heme

The Fe2+ porphyrin complex

Necessary, since no amino acid side chains can reversibly bind to oxygen

Oxygen only binds to the prosthetic group with iron in its reduced state

Prosthetic group

Independent group covalently bound to protein, it is necessary for protein function but not part of the protein.

Myoglobin structure

Single polypeptide chain with one heme group

One polypeptide chain with alpha helices

Only has tertiary structure because it only has one polypeptide chain

Hemoglobin structure

Four polypeptides with two identical alpha and two identical beta subunits

Has quaternary structure

Each alpha subunit and beta subunit can bind one oxygen

Heme binding

Oxygenation of myoglobin and hemoglobin is bound through six coordination bonds (not real bonds, learned in inorganic chemistry)

There are two critical histidine residues (proximal and distal)

Without oxygen bound, the heme group is puckered, when oxygen is bound, heme is planar

Deoxyhemoglobin is in the puckered state (unbound)

T (tense) state

Oxygen is unbound to heme

This is deoxygemoglobin

In the puckered state

R (relaxed) state

Oxygen is bound

This is oxyhemoglobin

This is in the planar state

Conversion from T to R state (helical representation)

Tyr42 bound to Asp99 →Asp94 bound to Asn

Both have hydrogen bonds for some stability because we need heme in both states

Concerted model of protein-ligand interactions

When four hemoglobin molecules are not bound (T-state) it favors the t state over the R state, when one bound it favors the T state still, if 2 is bound, it favors both because at half, t will favor empty and R will favor full. When 3 or 4 is bound, favors the r state.

Can be protein and substrate or heme picking up oxygen

Sequential models of protein-ligand interactions

This is a domino affect, when empty favors T, when one is bound it wants to bind more and the binding sites nearest to the bound ligand favor the R state, one still favors the T state, once 2 are bound it favors R completely because to two neighbors are changed to purple and favor the ligand binding and being in the R state of binding

Fractional saturation (theta)

The fraction of protein binding sites that are occupied

(Theta) = Occupied binding sites / total binding sites = [PL]/([PL] + [P])

[PL] is the concentration of protein-ligand complex

[P] is the concentration of protein

[L] is the concentration of ligand

Dissociation constant (Kd)

Opposite of fractional saturation

Reaction in which the dissociated species is the product

Kd = [P][L]/[PL]

When comparing two Kd values, a larger Kd indicates more of the dissociated species is present, and there is a lower affinity between the molecules

High Kd = low affinity

Low Kd = high affinity

How do you find the Kd on a fractional saturation curve

Follow the line until 50% fractional saturation, the x-value for the 50% point is the Kd

Compare a higher line on a sractional saturation curve to a lower line

The higher line has a lower Kd than the lower line Kd at 50% this means the higher line has a higher affinity because it has a lower Kd than the lower line.

Features of ligand-protein interactions

Ligand binding is a reversible process involving noncovalent interactions (ex. Coordinate bonds between O2 and Fe2+)

Ligand binding induces or stabilizes structural conformations in target proteins

Equilibrium between ligand-bound protein and ligand-free protein can be altered by the binding of effector molecules (effect the R vs T equilibrium) which can increase or decrease affinity

Bohr Effect

The relationship that pH and CO2 have on the ability of hemoglobin to bind to oxygen

there is a slight decrease in the localized pH due to the production of H+ from the equation CO2 + H2O → ← HCO3- + H+

Leads to a shift in fractional saturation

• Lower pH (ex. exercising) has a higher Kd (lower affinity)

• Normal pH is 7.4

• Higher pH (ex. hyperventilation) has a lower Kd (higher affinity)

2,3-BPG (2,3-Bisphosphoglycerate)

Found in RBC

Traps hemoglobin in the T state and acts as a negative effector

At higher elevations you have an increase in the 2,3-BPG in your blood so that you can increase the O2 release to their tissues (ultimately this increases the ATP production)

Maternal oxygen transport to fetus

Fetal hemoglobin has a lower affinity to bind to 2,3-BPG than regular

Fetus' need to be able to pick up the oxygen from it's mothers blood so that it can pick up oxygen when it is born (cant pick up oxygen nearly as well as adults)

2,3-BPG affinity facilitates the transfer of oxygen from the mother's hemoglobin to the fetus because fetal hemoglobin can obtain more oxygen if more hemoglobin molecules are in the R state

Anemia

Reduced oxygen transport efficiency from the lungs to the tissues

Altered hemoglobin function or reduced number of RBC

This occurs due to a mutation in alpha or beta hemoglobin subunits

Sickle cell anemia

Recessive genetic disease (advantageous against malaria when heterozygous, sickle cell when homozygous recessive)

Valine is substituted for glutamic acid at the 6 position in the beta-globin polypeptide (HbS)

A hydrophobic calapse is the result of the valine substitution because it is a smaller molecule than glutamic acid, which sickles the cell

Spleen turns over RBS quicker so the disease of malaria cannot spread as rapidly

Membrane transport proteins (three major classes)

Membrane receptors proteins

Membrane-bound metabolic enzymes

Membrane transport proteins

Membrane receptors proteins (major transport protein)

Involved in transducing extracellular signals across the plasma membrane

Membrane-bound metabolic enzymes (Major transport proteins)

Membrane proteins are embedded in the inner mitochondrial membrane, cloroplast thylakoid membrane

Membrane transport proteins (major class of transport protein)

Facilitates movement of polar molecules across the hydrophobic membrane

Membrane transport mechanisms

Biomolecules cross cell membranes in two fasions

• Hydrophobic molecules diffuse across lipids bilayers moving from a high to low concentration

• Polar molecules must be transported across cell membranes by membrane proteins to shield them from the nonpolar interior

Membrane transport proteins can fall into what two categories?

Passive: Facilitates biomolecule movement across a membrane in the same direction as the concentration gradient (does not require energy)

Active: Move biomolecules across the concentration gradient using energy

Porins

Porins frequently contain beta barrels

The primary sequence contains alternating polar and nonpolar residues (nonpolar residues face the hydrophobic region of the membrane, the hole in the beta barrels protein is lined with polar residues)

Binding sites for the substrate carrier proteins that pick up ions and small molecules are selective

Aquaporins

Can transport water, urea, or small molecules

A major class of passive membrane transport proteins

Transport water molecules across a hydrophobic membrane but can also transport urea and glycerol

11 different aquaporin genes that encodes a protein with 6 transmembrane alpha helices

Active membrane transport proteins

Require energy to “pump” molecules across the membrane

Two classes:

• Primary active

• Secondary active

Secondary active antiporter

Transport occurs in opposite directions

Red molecules move down their concentration gradient rives the green molecules up their concentration gradient

Primary active transport

ATP turnover drives the movement of molecules up their concentration gradient

Secondary active symporter

Transport occurs in the same direction

Red molecules move down their concentration gradient which drives the blue molecules up their concentration gradient

What are the two most abundant types of primary active transporters

P-type (phosphorylated)

ABC (ATP binding casset)

Both use ATP hydrolysis to drive large conformational changes in the protein complex

P-type transporters

Uses phosphorylation to drive protein conformational changes

Ex. Na+-K+ ATPase

• Integral membrane protein

• Exports 3 Na+ out of the cell for every two K+ ions imported into the cell

SERCA regulation

Phospholamban is coupled with the SERCA protein, at first it is an inactive complex (Ca2+ is in the cytosol)

Phospholamban is phosphorylated because epinepherine stimulates protein kinase A which results in phosphorylation

Once phospholamban is phosphorylated the Ca2+ channel on SERCA proteins open (this is when contraction happens)

ATP in the SERCA protein is closed from the cytosol, ATP is released as ADP and SERCA is phosphorylated releasing the Ca2+ into the sarcoplasmic reticulum lumen (muscles relax) ATP is added again to remove the pohsphorylated SERCA and reset the inactive complex

What is a domain of a protein

An area of the protein that has a specific function

What is the function of the SERCA N-domain

N domain is the area of SERCA protein that binds the nucleotide with ATP

What is the function of the SERCA P domain

The area of phosphorylation by hydrolysis of ATP utilizing Asp351

What is the function of the SERCA A domain

It is a stabilizing domain (stabilizes the SERCA N and P domains)

What is the function of the SERCA M domain

It is a transmembrane domain

What does phospholamban get phosphorylated by

Phosohprylation of Ser16 by protein kinase A

Phosphorylation of Thr17 by Ca2+/calmodulin kinase II

What does SERCA transport

Transports 2x Ca2+ ions into the sarcoplasmic reticulum

Ca2+-ATPase mechanism steps

1. ATP hydrolysis and phosphorylation of Asp351 releases H+ and binds Ca2+ in the transmembrane helices

2. ADP dissociates causing M2 to open towards the lumenal side and release Ca2+ (releases into the sarcoplasmic reticulum)

3. ATP binding repositions M2 to trap H+ (H+ are placeholders for Ca2+)

4. Asp351 dephosphorylates

Ca2+-ATPase mechanism is an example of a primary active transport

ABC Transporters

ATP binding cassettes

Act as ATP-dependent import and export proteins

ATP hydrolysis induces a large conformational change that converts the protein from outward facing to inward facing

Ex. Multidrug resistance protein (upregulated with taking certain drugs)

ABC transporter mechanism

1. Binding of the substrate carrier protein induces a conformational change to expose the substrate binding site to the periplasm side

2. ATP hydrolysis causes a conformational change that releases the substrate into the nucleotide binding domain

3. Release of substrate into the cytosol results in release of ADP and Pi and binding of ATP which resets the transporter

Secondary active transporters

Use energy available from downhill electrochemical gradient from one molecule to another to co-transport a second molecule against an uphill gradient

Transport is not coupled to ATP hydrolysis

Ex. Lactose permease

Lactose permease (secondary active transport)

Lactose is sealed from periplasm

Lactose transporter in inward conformations

Lactose can exit to cytoplasm

Mechanism is driven by the proton motive force (which is a symporter)

Titin is the largest protein in the body, how large

3 billion Dalton's

In muscle contractions which filaments move

Myosin is stationary, actin moves relative to myosin

Structure of muscle cells

Muscle cells are large fused cells (myoblasts) that contain nuclei and share sarcolemma (plasma membrane) and bundles of small fibers (myofibrils)

Myofibrils are composed of myosin and actin which are organized into thin and thick filaments

Myosin

Make up thick filaments that are arranged so that fibrous “tails” are associated in the middle and globular “heads” are at the ends

Actin

Make up thin filaments that are self-assembled

Tightly associated with tropomyosin, a second thin filaments protein

Actin and tropomyosin bind with troponin

Filament structure

Z-disk proteins attach to actin x2 and titin

Titin is attached to the myosin thick filaments

Actin houses troponyocin alpha-helices and troponin complex

What stimulates contraction of muscle filaments

1. In relaxed muscle, myosin binding sites on actin are blocked by tropomyosin

2. Ca2+ binding to tnC induces a conformational change in troponin and tropomyosin that uncovers the myosin binding site on actin

3. Myosin heads bind to actin and initiate muscle contraction

Calcium control and muscle contraction

1. Ca2+ binding to troponin uncovers myosin binding sites on actin thin filaments

2. Pi release induces the power stroke, which pulls the actin filament ~70 A towards the center

3. ADP release empties the nucleotide binding sites in myosin (relative movement of filaments causes muscle contraction)

4. ATP binding causes myosin to dissociated from actin

5. ATP hydrolysis induces the recovery conformations

What was the first enzyme to be crystallized and purified

Urease (pepsin)

Enzyme specificity

Lock and key (not likely)

• Substrate (reactants) binds to the enzyme perfectly

Induced fit

• Enzyme is flexible to accommodate the ill-fitting substrate

• Permits a much larger number of weaker interactions between the substrate and enzyme

Hexokinase ex as induced fit mechanism

Hexokinase catalyzes the first step in glycolysis

It also prevents unwanted side reactions due to the induced fit

Conformational selection mechanisms

Suggests that all conformations preexist, although ligand binding shifts the preference for a particular conformations

Once a primary conformations is “trapped” by ligand binding, induced fit may optimize the interactions

Interactions between the enzyme and substrate promote catalysis

Critical aspects of enzyme structure and function

1. Enzymes usually bind to substrates with high affinity and specificity

   • Active sites (binding pockets) in the enzyme bind to the substrate and promote catalytic reactions

2. Substrate binding to the active site induces changes in the enzyme

   • Ex. Hexokinase, a metabolic enzymes

   • Conformational change to block water from the active site and promote phosphorylation

3. Enzyme activity is highly regulated in cells

   • Modes of enzyme regulation:

     • Bioavailability (only found in specific places)

     • Catalytic efficiency (is it efficient)

   • pyridoxal phosphate is a coenzyme bound to the enzyme active site

   • Binding of AMP increases the catalytic efficiency of the enzyme

   • Phosphorylation of a ser residue also increases that catalytic efficiency of the enzyme

Enzymes are chemical catalysts that

Alter the rate of reaction without changing the ratio of substrates and products at equilibrium (only changes the Kinetics (rate))

Decreases the activation energy to speed up a reactions

Cofactors

Small molecules that aid in the catalytic reactions within the active site

Include inorganic ions such as Fe2+, Cu2+, and Mg2+

Coenzyme

Enzyme cofactors that require organic components

Include vitamin-derived species such as NAD+ and FAD

Prosthetic groups

Coenzymes that are permanently associated with enzymes (covalently attached)

Lipoamide as a coenzyme

Lipoamide is a coenzyme, a small group that is attached but not required for function of the protein

Enzyme classification system (EC)

1. Oxireductase - oxidation-reduction, transfer of H or O atoms

2. Transferase - transfer of functional groups Ex. Methyl, acyl, phosphoryl

3. Hydrolase - formation of two products by ydrolyzing a substrate

4. Lyase - cleavage of C-C, C-O, C-N, and other bonds by means other than hydrolysis or oxidation

5. Isomerase - Intramolecular rearrangements, transfer of groups within molecules

6. Ligase - Formation of C-C, C-O, C-S, or C-N bonds using ATP

Enzymes can increase the rate of reaction in three major ways;

1. Lower the activation energy by stabilizing the transition state

2. Providing an alternate path for product formation

3. Reduced entropy by orienting the substrates appropriately

Active sites contribute to catalytic properties how

1. Sequestered microenvironment of the active site.

   • Provides optimal orientation of the substrate relative to the reactive chemical group

   • Excludes excess solent

2. Binding interactions between the substrate and the enzyme to crease a transition state

3. Presence of catalytic functional groups

Transition state analogs

Stable molecules that mimic the proposed transition state

These molecules bing tightly to the active site

We usually don't isolate the transition state, a transition state analog drug can have a higher affinity than the actual transition state if it is more stable

Name the Catalytic functional groups (amino acids)

1. Hist

2. Asp, Glu

3. Ser, Thr

4. Tyr

5. Cys

6. Lys

7. Arg

Common catalytic mechanisms

1. Acid-base catalysis

   • Proton transfer that either involves water (specific acid-base) or a functional group (general acid-base)

2. Covalent catalysis

   • Nucleophile group on the enzyme attacks and electrophile center on the substrate to form a covalent enzyme-substrate intermediate

3. Metal ion catalysis

   • Metals are used to promote proper orientation of bound substrates and can aid in redox reactions

Enzyme-Mediated reactions

1. Coenzyme-dependent redox reactions

   • Include dehydrogenases

   • Involved NAD+/NADH, NADP+/NADPH, FAD/FADH2, and FMN/FMNH2

2. Metabolic transformation reactions

   • Involved isomerizations, condensations, and dehydration (hydrolysis) reactions

3. Reversible covalent modifications

   • Act as kmolecular switches by turning on/off cell signaling and gene expression

   • Include kinases and phosphatases

   • ATP is commonly used as a phosphoryl group source

Enzyme reaction mechanisms

Substrates bind to enzymes active sites through weak noncovalent interactions

Enzymes use conventional catalytic reactions mechanisms that follow basic principles of organic chemistry

Chymotrypsin (a serine protease)

Involves covalent and acid-base catalysis

Uses a catalytic triad (Hist, Asp, Ser) to form a hydrogen bonded network required for catalysis

Ser is converted to a highly reactive nucleophile

Catalytic mechanism for chymotrypsin

1. Polypeptide substrate binds to enzymes active site

   • Uses the catalytic triad

   • Has a substrate specificity pocket to know which AA to cleave

2. His57 removes a proton from Ser195 which allows a nucleophilic attack by the serine oxygen on the carbonyl carbon of the peptide

   • The molecules are cleaved on the C-terminal because fitting into the specificity pocket allows the correct orientation to cleave the AA

   • Serine becomes depronated in this step

3. His57 donates a proton to the amino group of the substrate, resulting in peptide bond cleavage. The carbonyl-terminal fragment is released as the first product

   • This kicks the amine out (with the rest of the peptide)

   • Oxyanion is stabilized in oxyanion hole made by backbone amino groups of Gly193 and Ser195

4. Water enters the active site His57 acts as a general base and removes a proton from water. The resulting OH- acts as a nucleophile and attacks the carbonyl carbon of the covalent acyl-enzyme intermediate

5. His57 donates a proton to Ser195, resulting in cleavage of the acyl-enzyme intermediate. The amino-terminal fragment is released as the second product and the catalytic triad is regenerated.

   • This step detaches the serin

6. The functional catalytic triad is regenerated within the enzyme active site

Chymotrypsin cleaves what

Cleaves the Aromatics

Large substrate binding pocket accommodates aromatic residues such as tyrosine