Biochem Lectures 10-19

Factor VII

An important enzyme in blood clotting. Responsible for cleavage for Factor X to Factor Xa. Mutations result in the most common genetic cause of hemophilia (blood fails to clot normally)

Interactions of proteins with membranes

1. Hydrophobic effect across bilayer (many) 2. Hydrophobic effect at bilayer surface (few) 3. Covalent lipid anchors 4. Via interaction with transmembrane (TM) proteins 5. Via ionic interactions with lipid surface

Structure of ATCase

12 polypeptide chains comprising of 6 catalytic and 6 regulatory subunits (2 catalytic trimers and 3 regulatory dimers) Binding CTP regulatory subunits triggers conformational change, controlling enzymatic activity.

PKA signaling

4 polypeptides PKA is normally inactive: forms a complex of two catalytic subunits (C) with two regulatory subunits (R) Regulatory subunits repress PKA activity cAMP binds the regulatory R subunits, leading to a conformational change: R subunits now dissociate off. The active sites of the catalytic C subunits are now freed (catalytically active kinase)

Lysosome

A low pH environment dedicated to proteolysis

Insulin

A peptide hormone that is generated by proteolytic processing. Proinsulin is cleaved to mature insulin (C peptide cleaved of, A chain and B chain remaining)

Glycogen phosphorylase

A target of cAMP-initiated signaling Glycogen phosphorylase (Mr 94.5 kDa): -expressed in liver and skeletal muscle -catalyzes this reaction glycogen (n glucose) to glycogen (n-1 glucose) with addition of Pi and release of glucose 1-phosphate. Glucose 1-phosphate goes to the liver to release glucose in the blood, and to the muscle for ATP synthesis.

Common polar membrane lipid head groups

Almost all are polar/charged Phosphatidylethanolamine - ethanolamine Phosphatidylcholine - choline Phosphatidylserine - serine Phosphatidylglycerol - glycerol Phosphatidylinositol 4,5-bisphosphate - myo-inositol 4,5-biphosphate (biological signals)

PKA

An enzyme activated by signaling Certain hormones (adrenaline for example) stimulate the production of a messenger molecule, cyclic AMP. cAMP in turn can activate protein kinase A (PKA) Phosphorylates Ser/Thr in its consensus sequence

Measurement of bilayer

About 50-80 A across 30A hydrophobic tails (15 per) 15A of outer leaflet

Kinetics of allosterically regulated enzymes

Activity frequently displays sigmoidal curves, plotting [S] vs. Vo The kinetics of allosteric regulations differ from Michaelis-Menten kinetics. Sigmoidal behavior makes activity switch-like and can be influenced by regulators.

Glycolipids

Another main class of membrane lipids in animals (based on sphingosine, different head) Head group is one or more sugars linked through glycosidic bonds. SEE DIAGRAM

Apmphiphiles

Aphiphilic compounds contain a polar head group and a nonpolar moiety (nonpolar tail)

ATCase

Aspartate Transcarbamoylase Allosteric feedback regulation Catalyzes early step in biosynthesis of pyrimidine nucleotides. ATCase is the first protein negatively regulated by CTP and positively regulated by ATP. The final product, CTP inhibits the first reaction catalyzed by ATCase

Cylindrical amphiphilic lipids

Assemble into membrane bilayers (liposomes) Two alkyl chains. Diameter of head group similar to that of hydrophobic moiety.

Biological membrane structure

Based on lipid bilayers Composed of lipids and membrane proteins Thin, fluid, fexible

FRAP

Bleach labeled lipids in plasma membrane with laser, then follow lipid diffusion as fluorescence recovery in the bleached spot (recovered fluorescence) Lipids diffuse rapidly within 1 monolayer (1um/s) which can be measured by FRAP.

Membrane proteins

Can be integral, penetrating hydrophobic core of membrane with hydrophobic amino acids or via lipid groups attached covalently through a cysteine (thioester/thioether) or through a GPI anchor.

Mechanisms for regulating enzyme amount

Cells use multiple mechanisms Allostery Covalent modification Proteolytic cleavage Local inhibitors Compartmentalization of enzymatic activity

GTPase

Cellular switches that can be found in GTP and GDP bound forms Example: Ras

Spacial and temporal control

Compartmentalizations can influence the access of enzymes to substrates. Lysosome, Nucleus Some proteins are only expressed at certain times, for example at specific stages of the cell cycle. Therefore only substrates that are present at the same time have an opportunity to be modified. (Depends on what's present at the same time)

Micelles

Conical/wedge shaped single chain amphiphiles (fatty acids, detergents); due to structure (most effecient way) Assemble into micelles at concentrations about their critical micellar concentration (CMC) Self assembly of nonpolar moieties lead to the shielding of their nonpolar surface from water; reduced effect.

Consensus sequence

Consensus sequence in a polypeptide defines amino acids as phosphorylation substrate for active PKA Phosphorylates Ser/Thr sidechains in proteins -X-R-(R/K)-X-(S/T)-B- Tyr kinases phosphorylates Tyr residues. Phosphorylation events reversible through protein phosphatase activity

Enzymatic activity and regulation

Controlling enzymatic activity is central to regulation Catalytic power of enzymes has to be adjusted to the fluctuating resources and needs of living cells.

Biological membrane functions

Define cell boundaries Define intracellular compartments Selectively permeable Maintain electric and chemical potentials and actively transport molecules and ions

Membranes

Define cells and most cellular compartments (compartmentalization) Lipid bilayers that are thin, fluid, flexible, and define cells + most cellular components Example: signaling across membranes is central to cancer. Integral membrane proteins involved

Unsaturated fatty acids

Double bonds in unsaturated fatty acids are almost always cis and rarely conjugated

Nucleus

Dynamic localization of proteins during cell cycle Example: the mitotic inducer cyclin-dependent kinase 1

Human blood groups

Glycolipids determine human blood groups (define the different blood-types A, B, AB, O) Antibodies act against shared blood. Differences in head groups (sugars)

Example of phosphorylations

PKA phosphorylates other proteins (covalent modification) This regulates the activity of other proteins, either positively or negatively cAMP - PKA - phosphorylase kinase - glycogen - phosphorylase pathway

Easily catalyzed

Energetically favorable, and ATP is highly concentrated inside cells.

Protein phosphatases

Ensure reversibility, a key feature of protein regulation. Reverses and returns phosphorylated protein to Ser/Thr/Tyr

Slow mechanisms of regulation?

Enzyme synthesis and proteolytic degradation are comparatively slow mechanisms for regulating enzyme concentration (frequently too slow to adequately regulate protein activity)

Regulatory domains

Enzymes frequently contain regulatory domains (turn on/off protein activity, control localization) Regulatory domains often control: -protein localization -substrate specificity -enzymatic activity -binding partners when protein is part of a larger complex

T/F: Phosphorylation is an ineffective modification.

FALSE Phosphoryl group adds two negative charges, and amino acids can form highly direction hydrogen bonds. This results in strong conformational changes in the phosphorylated target. One kinase can phosphorylate multiple target proteins. This results in signal amplification Energetically favorable, and ATP is highly concentrated inside cells. Thus, it's easily catalyzed

T/F: Membrane lipids are symmetrically distributed

False. Membrane lipids are asymmetrically distributed. Example: erythrocyte plasma membrane Membrane composition is asymmetrical, with different lipids on the outside than on the inside of the cell

Lipids

Fat Lipids are barely soluble in water but are soluble in organic solvents.

Structure of fatty acids

Fatty acids are unbranched carboxylic acids with C4 to C36 chains (saturated - no double bonds, or unsaturated) The conformation of fatty acids is determined by the degree of saturation of they alkyl chains.

Key idea behind ATCase

Feedback inhibition in a metabolic pathway Allosteric feedback regulation - one component of a pathway regulates another component of the same pathways

Lipids and proteins

Form membranes together. Each cell type has a specific composition of plasma membrane.

Testosterone

Forms estradiol Male and female sex hormones. Influence secondary sexual characteristics; regulate female reproductive cycle.

Triacylglycerol function

Function as storage lipids in fat droplets in adipocytes. -high energy content per weight -unhydrated, no solvation water to be carried (very efficient energy storage) -provide efficient energy storage and insulation.

Peptide hormones

Generated from precursors by proteolytic processing. Peptide hormones are synthesized prohormones. -packaged and stored in secretory granules (inside pancreatic cells) -processed by specific proteolysis before release -can bind to specific receptors on cells in other tissues.

Cortisol

Glucocorticoid Affects protein and carbohydrate metabolism; suppresses immune response, inflammation, and allergic responses.

Structure of membrain lipids

Glycerol backbone, linked to fatty acid by ester linkages.

Glycolipids

Head group is one or more sugars linked through glycosidic bonds.

Phosphatidylcholine molecular structure

Head group linked to choline. The fatty-acids aren't right next to each other; avoid resonance structure (break up with sp3)

Two general categories of phosphorylation

In eukaryotic cells 1. Phosphorylation of hydroxyl group of Ser/Thr 2. Tyr phosphorylation Ser, Thr, or Tyr residue have OH group. The OH group reacts with ATP, taking away a phosphoryl group.

Zymogens in the digestive system

In the duodenum, pancreatic zymogens meet their master switch: enteropeptidase. (Trypsinogen becomes trypsin which then activates multiple other enzymes) Upon proteolytic processing, zymogens undergo a conformational change: the active enzyme is generated

Lateral diffusion vs. spontaneous flip-flop

Lateral diffusion of lipids is common and rapid. A lipid molecule can diffuse around the cell within seconds (1um/s) Spontaneous flip-flop of lipids is a rare event. It's very slow to exchange between monolayers without protein catalysts (t1/2 in days). No solvent to shield the charges.

Blood clotting cascade

Initiated by platelets aggregating on damaged blood vessel surface. Enzymatic cascade of proteolytic processing. Prothrombin --> thrombin, fibrinogen --> fibrin, further crosslinking forms fibrin clots. Full cascade is more complicated.

Regulation by covalent modification

Kinases regulate target proteins by transferring phosphoryl groups. To fine-tune and control responses temporarily, modifications need to be reversible. Protein phosphotases are crucial: ensure reversibility, a key feature of protein regulation.

Summary of Lecture 10

Learn the principles of the principles of protein regulation. Learn some of the common themes of protein regulation. Learn how allostery can extend to enzymes Learn how covalent modification, especially phosphorylation, can regulate proteins. Learn how proteolytic cleavage can be used to regulate proteins. Learn the basics of GTPases and how they function as switches.

Signaling

Lipid bilayer composition can be used in signaling. PI3K can phosphorylate membrane lipids, producing PIP3. PIP3 is bound specifically by certain proteins (e.g. the PH domain of Akt kinase). Activation of Akt leads to activation of many growth pathways in the cell. PI3K phosphorylation can be reversed by PTEN phosphatase.

Other covalent modifications can regulate proteins

Methylation - reversible methylation of bacterial chemotaxis receptors by a methyl-transferase regulates flagellar movement ADP-ribosylation - catalyzed by cholera toxin, inhibiting an intracellular signaling pathway Adenylylation - adenylyl transferase regulates bacterial glutamine synthetase, inhibiting it by AMP modification

Assembly of amphiphilic lipids

Micelles and liposomes Driven together by the hydrophobic effect

Corticosterone

Mineralocorticoid Forms Aldosterone. Regulate reabsorption of Na+, Cl-, HCO3- in the kidney.

Naming fatty acids

Named by the number of carbons and the number+location of double bonds. Start with acid group and count out. In the systematic name, n refers to normal, straight chains with no branch. 18:1(D9) 1 = number of double bonds D9 = 1st side of double-bond (at the 9th C) Almost all are cis, and almost all have even C. The melting point increases as the mass increases (for saturated). Inclusion of double bond dramatically decreases melting temperature (change in fluidity)

Fatty acids

Natural, biological fatty acids have carboxylic acid attached to (generally unbranched) long tails. Amphiphiles

Triacylglycerol structure

Neutral lipids Contain 3 fatty acids in ester linkage with glycerol. (Mixture of different fatty acids)

Biological waxes

Neutral lipids. Waxes are esters of long-chain fatty acids with long-chain alcohols (C16-C30). Long-chain alcohol and a fatty acid connected via ester linkage They serve as water repellent and for protection.

PKA downstream targets

PKA may have downstream targets Hormones signaling and PKA regulation serves to regulate and coordinate multiple metabolic pathways. -both carbohydrates and lipid metabolism -note that it even regulates hormone production itself in a feedback loop.

Phosphatidate

One backbone structure of membrane lipids. Lipid backbone: diacylglycerol is phosphorylated at C3 of glycerol to form diacylglycerol 3-phosphate. This parent compound substituted with -H phosphatidate. Two fatty acids attached to glycerol and a head group with a phosphate that can be further linked to molecules like choline.

Signal amplification

One kinase can phosphorylate multiple target proteins.

Phospholipids

One of the main classes of membrane lipids in animals. Polar head group is an alcohol linked through a phosphodiester. Overall structure of phospholipids based on phosphatidate or acylated sphingosine is similar.

Phosphatidylcholine (PC)

Phosphatidate-based phospholipid.

Sphingomyelin

Phospholipid based on acylated sphingosine. Polar headgroup on sphingomyelin is phosphocholine.. Fatty acid is different Properties of phospholipids based on acylated sphingosine -head group X is phosphodiester-linked ethanolamine or choline, which introduces charges. -lipid backbone carries no net charge.

Three main classes of membrane lipids exist in animals

Phospholipids, glycolipids, sterols

Strong conformational changes

Phosphoryl group adds two negative charges, and amino acids can form highly direction hydrogen bonds.

PKA part of signal transduction pathway

Phosphorylase kinase is: -a substrate of PKA -an enzyme that phosphorylates and activates glycogen phosphorylase, a key enzyme of glycogen metabolism -part of the cAMP signal transduction pathway that regulates glycogen metabolism. Increase in cAMP increases PKA, which increases phosphorylase kinase, which increases glycogen phosphorylase

Phospholipids

Polar head group is an alcohol linked through a phosphodiester

Regulation at a Protein Level

Principles of protein regulation Regulation y reversible ligan binding Regulation by covalent modification Irreversible protein regulation by proteolysis Regulation within cells by compartmentalization

Lipid diffusion

Probing lipid diffusion with labeled lipids. Experimental approach using live cells: FRAP (fluorescence recovery after photobleaching)

Florescent labels

Probing lipids with florescent labels. Electrophilic and aromatic substitution Lipids can be labeled with a fluorophore. Example is TNBS, which react with PE

Zymogen

Proenzyme An inactive enzyme precursor activated by proteolytic cleavage

Structural function of cholestrol

Proper regulation of membrane fluidity, modulates protein membranes. Breaks up van der Waals interactions and close packing of the phospholipid alkyl chains. This maintains lipid bilayer in fluid phase even when thermal motion is low. At normal temperature, it sterically hinders lipid diffusion.

Target proteins

Proproteins Functional digestive enzymes Hormone precursors Cellular proteins for programmed cell death (apoptosis) Structural proteins (e.g. blood clotting or collagen maturation)

Response to signals

Protein kinases can respond to signals Input --> protein kinase --> phosphorylated proteins Some examples of input signals: -cyclic nucleotides (cAMP/PKA) -Ca2+ and calmodulin -diacylglycerol -other phosphoproteins

Blood clotting

Proteolytic cleavage triggers blood clotting Blood clotting is executed by a series of proteolytic steps: each steps activates a proprotein. Central to clotting is one of the cleavage of fibrinogen by thrombin produces fibrin.

Phase transitions

Pure lipid bilayers undergo phase transitions. Heat increases thermal motion: no regular array of lipids as their alkyl chains become less ordered, bilayer attains fluid-state properties. Fluidity is necessary for membranes to function. At low temperatures, it's in a paracrystalline state (gel) - short/medium range ordering in lattice. Membranes are fluid at physiological temperatures, but can form a paracrystalline state at low temperatures

Critical micellar concentration

Solubility of amphiphilic monomers: -as concentration is raised, their solubility limit is reached. -at that concentration the critical micellar concentration (cmc) amphiphilic monomers self assemble into micelles SEE GRAPH

Regulation of enzyme amount

Regulated gene expression (on/off) Protein degradation

Phosphorlylation

Regulation by covalent modification. Phosphoryl groups can engage in hydrogen bonds and ionic interactions. Phosphoryl groups can change the conformation of the modified proteins. This can lead to activation or inactivation depending on the modified protein.

Pancreatic zymogen

Released from pancreatic cells through secretory pathway via zymogen granules

Acylated sphingosine

Second key membrane lipid backbone. A long chain amino alcohol. Linked to one acyl group acylated sphingosine substituted with -H : ceramide Backbone is continuous. Have same headgroup (phosphate-linked to choline) to make sphingomyelin.

ATCase sigmoid reaction velocity curve

Sigmoidal Vo plot is not Michael-Menten behavior, but still demonstrates saturation. (1/2)Vmax-K0.5

Cholesterol

Small polar head group: C-3 hydroxyl Precursor for many hormones (cortisol, testosterone, and estradiol) Very hydrophobic

Specificity of protein kinase

Some may recognize only a single substrate More commonly, kinases recognize consensus motifs present in multiple substrates. Other domains or subunits outside the catalytic domain can contribute to specificity Temporal and spacial control can also influence which substrates are phosphorylated.

Integral proteins

Span across the membrane. Hard to extract without powerful detergents.

Sterols

Steroid nucleus with one polar hydroxyl group

GTP hydrolysis

Stimulated by GTPase activator proteins (GAPs) Factors that help the G-protein return to the GTP-bound state are called GTP-GDP exchange factors (GEFs). Hydrolysis of GTP assisted by GTPase activator proteins (GAPs) and the exchange of GDP --> GTP is assisted by GTP exchange factors (GEFs)

Packing into membrane bilayers

Structures of polar lipids facilitate their packing into membrane bilayers.

G-proteins

Switchable signaling proteins G-proteins bind and hydrolyze GTP. This allows them to cycle between conformational states.

Structure of catalytic subunit

The catalytic groove Peptide, phosphorylation site is at Ser The inactive complex: when the regulatory subunit inserts itself into the groove

Amphiphilic molecules in aqueous solution

The nonpolar moiety is subject to the hydrophobic effect as it orders surrounding water molecules and decreases entropy.

T/F: Enzymes can also have multiple polypeptides, especially in cases with allostery

True. Typically multi-subunit proteins Cooperativity binding between multiple binding sites Modulators induce a conformational T --> R transition Sigmoid curve of binding or reaction velocity

Trypsin and trypsinogen

Trypsinogen (inactive) gets cleaved before the 7th amino acid isoleucine to become trypsin (active) Note: this irreversible activation step calls for special protection of the cells secreting trysinogen

Proteolytic cleavage

Used both intra- and extracellularly Irreversible, needs to be controlled tightly Can activate or inactivate target proteins (protein destruction = negatively, removal of propeptides = positively) Essentially irreversible, other protections used to regulate proteases (compartmentalization and local inhibition)

Pancreatic trypsin inhibitor (PTI)

Zymogen granules contain PTI (local inhibitor) to safeguard against trypsin activity. PTI binds to trypsin very tightly. Inserts a loop resembling a trypsin substrate into the catalytic cleft to inhibit (competitive inhibition?)

Precursor for many hormones

cholesterol is a precursor for all steroid hormones in humans. Formed by side-chain cleavage and oxidation of sterol rings. Synthesis of steroid hormones occurs most prominently in adrenal glands and gonads.