BCH110A Lectures 9-18 Learning Objectives/Key Concepts
Describe the acyl-enzyme intermediate and identify the type of bond attaching the acyl group to the enzyme (Is it an amide linkage? anhydride? ester? etc.). Describe how that acyl group relates to the structure of the original substrate.
Acyl-enzyme intermediate is a covalent transient molecule; formed by the reformation of the C=O double bond of the tetrahedral intermediate. Ester bond formed towards the Ser hydroxyl group. Acyl-enzyme intermediate releases product and remains in the transition state until another substrate binds and creates the second product, where the enzyme can revert back to original or keep the cycle going.
Indicate what is being acylated and deacylated in the chymotrypsin reaction (be specific about the functional group involved)
Acylation - active site Ser attacks carbonyl carbon of the substrate, resulting in cleavage of the peptide(or ester) bond, releasing of the C-terminal part of substrate and formation of acyl-enzyme intermediate with N-terminal part of residue Deacylation - water molecule takes the place of the Ser side chain in active site; acyl-enzyme ester bond hydrolyzed and releases C-terminal half of substrate.
Explain the role of the "oxyanion hole" in the mechanism
Aids in creating the acyl-enzyme intermediate and stabilizes the tetrahedral intermediate in the proteolysis reaction. Contains H-bond donors to interact with the negative charge of oxygen(oxyanion), stabilizes the tetrahedral intermediate, H-bonding interactions with backbone NH groups from the protein at a site(oxyanion hole), residues are already in correct location and orientation to form two strong H-bonds, NOT induced fit
Describe the general structure of protein kinases in terms of 2 lobes, their ATP binding and substrate binding, and how that structure relates to their regulation, including the roles of the activation loop and the P loop.
All protein kinases have 2 lobes enclosing active site: "P Loop" that helps align and bind phosphates of ATP substrate "activation loop" that blocks the substrate binding site two lobes of the kinase close on the active site and the C-terminal C helix moves, creating a binding cleft for the target. The activation loop moves away, allowing the catalytic loop to swing into place A conformational change moving the A loop activates a protein kinase
What are 5 ways to regulate protein activity?
Allosteric Control Multiple forms of enzymes(isozymes) Interaction with regulatory proteins Covalent Modification Proteolytic Activation
Name 2 types of secondary structural motifs/elements used by integral membrane proteins to cross membranes. Describe where the R groups are located in these secondary structural elements relative to the hydrophobic lipid core.
Alpha-helical or beta-barrel motifs Glycophorin A Bacteriorhodopsin Porins The hydrophobic side chains point out from the alpha-helix and screen the polar backbone(carbonyl oxygens and amide N-H groups)
Identify the leaving group coming from each of the tetrahedral intermediates as the intermediate breaks down.
Amine group leaves the enzyme and binds to Serine(from the reformation of a double bond with carbon which breaks the peptide bond between the carbon and amino acid group) In deacylation, collapse of the tetrahedral intermediate forms a carboxylate anion, and displaces Ser 195. Proton from histidine returns to Serine. Carboxylic acid released.
With the structure of a lipid as an example, point out the features that make a molecule amphipathic.
Amphipathic molecules have two distinct ends: a hydrophilic AND hydrophobic part. Fatty acids, components of storage lipids(triacylglycerols), are long-chain carboxylic acids that have a hydrophilic RCOO- head group and a hydrophobic tail.
Lipid-Anchored Proteins
Amphitrophic proteins associate reversibly with the membrane, usually in a regulated way and often involving reversibly attached lipid anchors or conformational changes in the protein
Explain the role of each member of the catalytic triad in the reaction.
Asp 102: positions His 57 in the correct orientation so the His and the Ser interaction can occur His 57: nitrogen with partial negative charge will interact H(partial positive) on Ser Ser 195: residue that acts as a nucleophile that attacks the carbonyl carbon The serine has an -OH group that is able to act as a nucleophile, attacking the carbonyl carbon of the peptide bond of the substrate(covalent catalysis). A pair of electrons on the histidine nitrogen has the ability to accept the hydrogen from the serine -OH group, thus coordinating the attack of the peptide bond(acid/base catalysis) The carboxyl group on the aspartic acid coordinates with the histidine, making the nitrogen atom more electronegative through desolvation.
Draw structure of the catalytic triad at the beginning of the reaction, and explain how the ionization states and hydrogen bonding pattern of these three residues change step by step during catalysis.
Asp pulls on His, making it a better base His pulls on Ser, just enough to align it so the target substrate can deprotonate it briefly to form an alkoxide ion to be a nucleophile Converts OH group of Ser 195 into a potent nucleophile, and then also converts O of H2O into a potent nucleophile
Explain how hexokinase increases the nucleophilicity of glucose's 6-OH to attack the g-phosphate of ATP, and the role of Mg2+ in the reaction
Basic group(H sticking off of D-glucose) on enzyme accepts proton from the sugar(general acid base catalysis), making sugar a stronger nucleophile. O: more negatively charged, so it attacks the phosphate group Magnesium(Mg2+) helps reduce the negative charge of the oxygens; allows hydroxyl group to attack much easier
Peripheral Membrane Proteins
Bind to the membrane surface through electrostatic interactions(ionic & hydrogen bonds with polar lipid head groups) or with integral membrane proteins. They are removable by treatment with high salt or change in pH.
Describe substrate binding, including the role and chemical nature of the "specificity pocket" in chymotrypsin, and which peptide bond in the substrate (relative to the specificity group) will be cleaved.
Binding of enzyme to substrate yields ES complex. The transition state binds to enzyme tighter than enzyme binds to the substrate(if bound to substrate tighter, it raises activation energy as more energy needed to make product). Binding occurs at the enzyme active site(excludes water). Binding uses H-bonds, salt bridges, van der Waals, hydrophobic effect to bind active site with substrate(all weak). Once enzyme binds favorably to transition state, induced fit makes active site complementary to transition state(lowers free energy of transition state, lower barrier, higher rate). The hydrophobic pocket in chymotrypsin attracts large hydrophobic side chains into the pocket, aligning the carboxyl group right over the Ser hydroxyl group, which attacks the positively charged carbon and breaks the amide bond. Cleaves the peptide sequence right after the large hydrophobic residues
How does electrostatic catalysis(including desolvation) enhance reaction rates?
By removing water from active site(desolvation), the protein stabilizes a dipole moment or makes it stronger by bringing a charge closer to it(changes how the electrons are distributed in a bond) Lower dielectric constant environment than H2O(more nonpolar environment), stronger electrostatic interactions(strength inversely related to dielectric constant)
Briefly explain the consequences if an individual has a genetic deficiency in any one specific enzyme involved in glycosphingolipid degradation.
Can result in abnormal accunulation of partially degraded lipids, with toxic results(genetic diseases) Tay-Sachs disease is due to lack of hexosaminidase A, which is needed to hydrolyze the glycosidic bond attaching terminal N-acetylgalactosamine residue in glycosphingolipid GM2; results in mental retardation, blindness, muscular weakness, paralysis and death by age 3-4 Niemann-Pick disease is due to the lack of sphingomyelinase, which hydrolyzes the phosphate ester linkage of phosphocholine to ceramide. Symptoms include enlarged liver and spleen, mental retardation, early death
Explain how a cascade of catalysts (e.g., in PKA activation, or in blood clotting) results in amplification of a signal.
Cascade produces rapid and enormous amplification of original signal because every activated enzyme molecule can itself catalyze conversion of many substrates first event triggered by some signal that initiates cascade, e.g. a hormone binding to a receptor, or a wound triggering the blood clotting cascade Cascade: a series of events in which each event in series is catalyzed by an enzyme activated in previous event
What is the difference between kinetic and thermodynamic stability?
Chymotrypsin: two phase kinetics; pre-steady state and steady-state Kinetic stability: a measure of how rapidly a protein unfolds; structure does not change much over time Thermodynamic stability: when a system is in its lowest energy state, or chemical equilibrium
Briefly describe the structural change that occurs upon the activation of chymotrypsinogen, including what changes occur in the active site.
Chymotrypsinogen is activated to chymotrypsin in a sequence of endoproteolytic steps: peptide bond between Lys15 and Ile16 is cleaved by Trypsin, generating new alpha-amino group at Ile16. The new alpha-amine turns inward and makes a salt bridge with Asp194 stabilizing the spatial arrangement of Ser195 and with it induces formation of the catalytic triad of the active site. The second proteolytic step further stabilized this active configuration.
How does induced fit increase the rate of chemical reactions?
Conformational changes resulting from substrate binding; enzymes induce conformational change so substrate can fit nicely to enzyme; binding can stabilize different conformations of enzymes or substrate or both orients catalytic groups on enzymes which promotes tighter transition state binding, and/or excludes H2O
How does covalent catalysis enhance reaction rates?
Covalent catalysis enhances the rate by the transient formation of a covalent catalyst-substrate bond Product from formation is a covalent intermediate that is used in the next step, which lowers the activation energy compared to a non-covalent catalytic mechanism - enzyme alters the pathway to get to the product
How do the three other classes of proteases (besides the serine proteases) generate nucleophiles potent enough to attack a peptide carbonyl group?
Cys proteases: nucleophile is Cys thiol activated by His(gen. base) Asp proteases: nucleophile is HOH itself assisted by 2 Asp residues: general base catalysis by 1 Asp carboxyl group and orientation/polarization of substrate carbonyl by 2nd Asp residue Metalloproteases: nucleophile is HOH assisted by binding to a metal(e.g., Zn2+) and by general base catalysis by some enzyme base group
Know the definitions of: proteolysis, serine protease, nucleophile, leaving group, general acid, general base, catalytic triad, acyl group, tetrahedral intermediate, acyl-enzyme intermediate, acylation, deacylation, oxyanion hole, induced fit, SN2
Definitions
Explain what determines A, B, O blood groups (no detailed structures).
Determined by the pattern of carbohydrates on glycosphingolipids (and also some glycoproteins) on the outer side of cell membranes Specific enzymes are required to add each specific sugar residue in the right order in the right linkage Type O have no functional transferase activity. Type A transferase adds N-acetylgalactosamine(GalNAc) Type B transferase adds galactose(Gal) Type AB individuals have both enzymes, so have both antigens
What is the starting fatty acid for synthesis of the eicosanoids, such as prostaglandin, and where does it come from? How do non-steroidal anti-inflammatory drugs (NSAIDs) reduce inflammation? How do steroids reduce inflammation?
Eicosanoids: Prostaglandins(mediate fever), thromboxanes(blood clotting), leukotrienes(smooth muscle contraction, overproduction causes asthmatic attacks) Eicosanoids are paracrine hormones(locally acting) that are all synthesized from arachidonic acid, removed by PLA2 from position 2 of the membrane glycerophospholipids; steroids inhibit PLA2 and thus inflammation. Non-steroidal anti-inflammatory drugs like ibuprofen block the substrate channel and inhibit the enzyme, preventing prostaglandin synthesis and reducing inflammation
Describe the covalent enzyme intermediate (altered enzyme form) with a high negative free energy of hydrolysis that is used to "store" some of the potential energy from the oxidation reaction, to allow the condensation of the carboxylic acid group of 3-phosphoglycerate with phosphoric acid (inorganic phosphate) to produce an acyl phosphate, a mixed anhydride with a high negative free energy of hydrolysis.
Energy released by carbon oxidation is converted into high phosphoryl-transfer potential. The favorable oxidation and unfavorable phosphorylation reactions are coupled by the thioester intermediate, which preserves much of the free energy released in the oxidation reaction. The use of a covalent enzyme-bound intermediate as a mechanism of energy coupling.
Describe the general mechanism by which zymogens are activated.
Enteropeptidase, an enzyme secreted by cells that line the duodenum(small intestine), activates a small amount of trypsinogen to trypsin, which coordinates control of zymogen activation outside cells The zymogen form is chymotrypsinogen synthesized in the pancreatic acinar cells and secreted into the digestive tract. There the chymotrypsinogen is activated to chymotrypsin in a sequence of endoproteolytic steps.
What are the 2 enzymes involved in glycogen metabolism whose activities are reciprocally regulated by phosphorylation and dephosphorylation?
Glycogen phosphorylase: breaks down glycogen when you need to get glucose out of "storage" Glycogen synthase: synthesizes glycogen when you have excess glucose and need to store it away the cell doesn't "want" these 2 opposing enzymes to be active under the same conditions, so they're regulated reciprocally BOTH enzymes phosphorylated when cells wants to get glucose out of storage -Glycogen phosphorylase b: the phosphorylated form is more active -Glycogen synthase b: the phosphorylated form is less active -they aren't both working in the cell at the same time, result of reciprocal regulation
Discuss the structural properties of the following examples of membrane proteins: glycophorin A, bacteriorhodopsin, a porin, and prostaglandin H2 synthase. Include in your discussion the type(s) of secondary structure and types of R groups found in the components in contact with hydrophobic core.
Glycophorin A - 19-20 AA stretches of hydrophobic residues are very common way that proteins span biological membranes - in alpha-helical conformation; alpha-helix spans hydrophobic core of membrane Bacteriorhodopsin - 7 transmembrane alpha-helices; generates a proton gradient that is used to drive ATP synthesis; amphipathic alpha-helices have hydrophobic and hydrophilic residues are spaced such that one side of the helix is hydrophobic and one side is hydrophilic. The hydrophilic sides stack together on the inside of the folded protein, away from the membrane. The light-absorbing cofactor retinal binds in the center, and with a hydrogen-bonding network in the hydrophilic residues that work in proton translocation Porin - quaternary structure(homotrimers); channel forming proteins consisting of large 16-18 strand antiparallel beta-sheet that folds into a cylinder(beta-barrel).Again, the polar peptide backbone groups (carbonyl O and amide N-H groups) are fully hydrogen-bonded in the membrane-integral core of membrane in the beta barrel secondary structure. As with the amphipathic alpha helix, the outward facing amino acid residues are hydrophobic and the inward facing residues hydrophilic... In this case, however, the inward facing residues form a water-filled channel that penetrates the membrane. H2 synthase(prostaglandin): on surface of ER membrane but anchored in membrane by a set of alpha-helices with hydrophobic R groups that extend into membrane core
Identify the nucleophile that attacks the carbonyl carbon in both the acylation and deacylation steps of the catalytic mechanism.
In acylation, the active site Ser attacks the carbonyl carbon of the substrate, resulting in cleavage of the peptide( or ester) bond, results in release of the C-terminal part of substrate and formation of covalent acyl-enzyme intermediate with N-terminal part of residue In deacylation, water binds to active site in position formerly occupied by Ser 195, adjacent to His 57. Same mechanism is used to activate the water molecule that was used to activate hydroxyl group of the Serine. Resulting hydroxide undertakes nucleophilic attack on carbonyl carbon of acyl-enzyme intermediate, forming second tetrahedral intermediate.
Principles by which enzymes enhance reaction rates include:
Induced Fit Proximity and Orientation General acid/base Catalysis Covalent Catalysis Electrostatic Catalysis(including desolvation) Strain(preferential binding of the transition state)
Explain in structural terms how an integral membrane protein can deal with its polar backbone groups in spanning the hydrophobic core of a lipid bilayer.
Integral membrane protein accommodate their polar backbone (peptide N-Hand C=O groups) in a stable way across the hydrophobic core of the lipid bilayer by hydrogen-bonding as many as possible of polar backbone groups(really ALL of them), i.e., by forming secondary structures, either alpha-helical or beta-barrel motifs for the membrane-spanning parts of the protein.
Explain the biological usefulness of isozymes, and discuss the example of muscle hexokinase vs. liver glucokinase in terms of difference in function of the tissues.
Isozymes - multiple forms of an enzyme that catalyze the same reaction, but with different kinetic and regulatory properties hexokinase(muscle) - muscle function = contraction, breaks down glucose for energy; gets glucose from blood glucokinase(liver) - maintenance of blood glucose at 4-5mM, liver takes up and stores excess glucose and exports it, as needed Function of hexokinase/glucokinase: glucose entering cells from blood is phosphorylated, trapping it inside Kinetic properties of hexokinase I(muscle) and hexokinase IV(glucokinase, liver) fit different metabolic needs in liver and muscle
Explain the dependence of kcat on pH.
Kcat refers to enzyme specificity(how fast the enzyme turns over). As pH increases, Kcat increases. In basic environments, Kcat is higher. In acidic environments, Kcat is lower. Histidine must be unprotonated for good catalysis, higher pH means lower [H+] concentration
How do components within the membrane move?
Lateral diffusion: rapid for both proteins and lipids within the plane of the membrane(except for anchored proteins, for example to cytoskeleton) Transverse diffusion: "flip-flop" of both proteins and lipids is extremely low, unless mediated by protein "flippases" Lipid composition in the 2 leaflets of bilayer is asymmetric, as is protein distribution
For charged solutes the presence of a membrane potential as well as the chemical concentration gradient influences the distribution of the solute, what equation shows this?
Mechanisms of transport processes involving membranes usually involve protein conformational changes.
Understand the normal terminology for shorthand nomenclature for fatty acids, e.g., 18:3(D9,12,15) and also the "w" terminology.
Number the carbons starting from 1; 2 is alpha carbon omega (w) C, starting from terminal CH3 group, w is 1; omega # fatty acid depends on where the double bond starts
How does orientation enhance reaction rates?
Orientation of the reactants can increase reaction rates IF oriented optimally
What is being oxidized and reduced in the glyceraldehyde 3-phosphate dehydrogenase reaction?
Oxidized: glyceraldehyde 3-phosphate Reduced: NAD+ to NADH
What bond in a glycerophospholipid is cleaved (hydrolyzed) by phospholipase A1? A2? C? D?
PLA1 cleaves ester bond to C1 OH PLA2 cleaves ester bond to C2 OH PLC cleaves phosphate ester bond to C3 OH PLD cleaves phosphate ester bond to "X" (other alcohol on C3 phosphate)
Discuss the protective mechanism that keeps prematurely activated pancreatic digestive enzymes inside the acinar cells from autodigesting the pancreas, and describe/name an example.
Pancreatic Trypsin Inhibitor, PTI, is a small, very specific, very tight-binding inhibitor of trypsin. It inhibits any trypsinogen molecule that's accidentally activated prematurely. Combination of very tight binding and very slow catalytic turnover makes PTI a very effective inhibitor. Trypsin initiates extracellular activation of all the pancreatic zymogens.
Describe the permeability properties of lipid bilayers.
Permeability coefficients correlate with solubility in nonpolar solvent relative to solubility in H2O. Membranes are highly impermeable to ions and most polar molecules, but more permeable to nonpolar species. Lipids diffuse freely(mostly) laterally in the plane of the bilayer transverse diffusion of lipids between the layers (flip-flop) is slow, due to the high energy barrier of moving polar head group through the hydrophobic phase; can be catalyzed by specific integral membrane proteins called flippases, floppases, or scramblases, which usually require the input of free energy(hydrolysis of ATP)
List the biological roles and the molecular components of membranes.
Phospholipids, Glycolipids, Cholesterol(steroid): three principal classes of membrane lipids two principal types of lipid backbone: glycerol(glycerophospholipids), sphingosine(sphingolipids, both sphingophospholipids and sphingoglycolipids) ROLES OF MEMBRANES: keep toxic substances out(keeping wanted substances inside), allow specific molecules in,
Name ("generic" names) the types of enzymes that catalyze a) phosphorylation, and b) dephosphorylation of proteins; specify what types of amino acid functional groups are generally the targets of phosphorylation; and draw the structure of such enzyme functional groups before and after phosphorylation.
Phosphorylation - catalyzed by protein kinases(Serine/Threonine kinases, Tyrosine kinase) Dephosphorylation - catalyzed by protein phosphatases
Draw the general structure of a glycerophospholipid, and be able to recognize the specific structures of phosphatidic acid, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, and phosphatidyl inositol.
Polar head group attached to a glycerol backbone and up to two fatty acyl chains
What is the role of the coenzyme NAD+ (acting like a "cosubstrate") in the glyceraldehyde 3-phosphate dehydrogenase reaction?
Post-formation of the thiohemiacetal intermediate, oxidation to thioester intermediate(NAD+ to NADH) Carbon loses bound H to NAD+, in exchange the oxygen is activated by histidine, forming double bond between C and O NADH then converted back to NAD+; attack on thioester by phosphate group
Explain whether the dephosphorylation reaction is actually the chemical reverse of the phosphorylation reaction, and if not, what type of reaction the dephosphorylation represents.
Protein dephosphorylation is NOT the reverse of protein catalyzed phosphorylation reaction. Both reactions are irreversible. (Active) catalyst (kinase or phosphatase) needed for significant reaction rates, so the cell's "decision" about what fraction of target protein is phosphorylated vs dephosphorylated depends on how active the specific protein kinase is vs. how active the specific protein phosphatase is. Dephosphorylation(hydrolytic removal of the phosphate groups) catalyzed by protein phosphatases. The phosphate group is removed by hydrolysis of the phosphate ester(transfer of phosphate to H2O)
The chemical mechanism of serine proteases like chymotrypsin illustrates:
Proximity and Orientation Transition State Stabilization Induced Fit Covalent Catalysis(catalytic triad of Asp, His, and Ser) General Acid-Base Catalysis Electrostatic Catalysis
Sketch plots of vo vs. [S] for an allosteric enzyme that illustrate positive homotropic regulation and positive and negative heterotropic regulation, with ATCase as an example. Specifically, sketch (all on the same axes) for ATCase: vo vs. [aspartate] curves with no heterotropic regulators present, with an allosteric inhibitor present, and with an allosteric activator present.
-homotropic effector = activator (substrate aspartate) -heterotropic effectors (allosteric inhibitor = CTP; allosteric activator = ATP) No CTP or ATP - black With CTP - blue With ATP - red
Bacteriorhodopsin(membrane protein)
7 transmembrane helices
How does proximity enhance reaction rates?
Affects entropy; if enzyme and substrate are in close proximity at the active site, they are more likely to interact than if they were further apart(enzyme increases local concentration of reactants)
How does general acid/base catalysis enhance reaction rates?
Bronsted acid/bases help the enzyme avoid unstable charged intermediates in reaction.(a group that donates a proton[general acid] in catalysis has to accept a proton[general base] later in catalytic mechanisms for the catalyst to be regenerated in its original conjugate acid form
Write out the structure of a 16-carbon saturated fatty acid (i.e., no double bonds), and describe the general properties of the fatty acyl components of membrane lipids.
CH3(CH2)14COOH or C16H32O2 Longer and/or more saturated FA's are less water-soluble Longer and/or more saturated FA's have a higher melting point The number of double bonds affect melting point and fluidity because there are many van der Waals packing interactions between saturated chains that are disrupted by the bent cis double bonds of unsaturated fatty acids
Recognize the structure of cholesterol. Name two other categories of steroid compounds that are synthesized from cholesterol.
Cholesterol consists of four fused hydrocarbon rings(A-D), three with 6 carbons, the fourth with five(steroid nucleus)
How does interactions with regulatory proteins regulate protein activity?
Example: Calmodulin (CaM), a Ca2+ "sensing" protein - With Ca2+ bound, Ca2+-CaM binds to a variety of enzymes in the cell ("target enzymes"), regulating their activities, including Ca2+-calmodulin-dependent kinases (CaM kinases).
To what aspect do solutes move spontaneously?
From compartment of higher concentration to compartment of lower concentration.
Describe which type(s) of general catalytic principles (first learning objective above) are used by chymotrypsin, and how
Proximity and orientation - close and oriented so reaction can occur(between enzyme and substrates) Transition state stabilization - negative charge produced on the oxygen, moves into an environment where we have extra electrostatic interactions, stabilizing transition state Induced fit - pac man, substrate binding induces conformational change, removes water and positions active site residues properly Covalent catalysis, involving a "catalytic triad" of Asp, His and Ser in the active site - amide to ester to carboxyl and amines general acid-base catalysis - histidine abstracting a proton was able to absorb the proton, becoming acidic, remove the amine group and protonate(histidine acts as the acid) electrostatic catalysis(desolvation) - active sites gets reactants out of H2O, no water in active site(strong electric forces within), changes how electrons are distributed in a bond
What is the pKa of the Cys residue in the glyceraldehyde 3-phosphate dehydrogenase reaction? When NAD+ is in the active site? Would you expect G3P to be more active at pH 5 or pH 7?
I would expect the G3P to be more active at pH5 as there are more [H+] ions in solution to form NADH from NAD+
What type of proteases are an example of convergent and divergent evolution?
Serine proteases
What can hydropathy plots be used to predict?
The segments of primary structure of a membrane protein that might be transmembrane alpha-helices.
What regulates the amounts of many key enzymes?
Transcription, mRNA processing, and/or translation, destruction(proteolytic degradation) of old/unwanted enzymes, metabolic needs/conditions
Major functions of lipids:
energy storage, major membrane components; other lipids function as signals, electron carriers, emulsifying agents, hormones...
Porins(membrane protein)
large beta-beta barrel with aqueous channel down the center
How do isozymes regulate protein activity?
- Multiple forms of an enzyme that catalyze the same reaction - Different kinetic parameters like Km, and/or different allosteric regulation, with physiological consequences -Hexokinase -- different forms in liver vs. muscle reflect the different roles of those tissues in the bod
What secondary structures do integral membranes assume to get polar groups of polypeptide backbone across hydrophobic core of lipid bilayer?
-Membrane spanning alpha-helices -antiparallel beta-sheets wrapped into beta-barrels
How does allosteric control regulate protein activity?
-conformational changes(2 conformations in equilibrium, "R"(more active) and "T"(less active) -allosteric activators(positive effectors/modulators) -allosteric inhibitors(negative effectors/modulators) -feedback inhibitors(product of pathway inhibits first committed step in pathway) -homotropic effector -heterotropic effectors
Briefly explain the allosteric regulation of ATCase, including its quaternary structure, its role in metabolism, and how its activity is regulated by allosteric inhibition and activation. Include the physiological rationale for the inhibition and activation.
-homotropic effector = activator (substrate aspartate) -heterotropic effectors (allosteric inhibitor = CTP; allosteric activator = ATP) -CTP binds preferentially to T state, thus stabilizing T state, shifts R-T equilibrium toward T state, so Vo vs. [S] curve shifts to the right -ATP preferentially binds to R state -> Vo vs. [S] curve shifts to left -binding of substrate to one active site in a molecule increases likelihood that the enzyme will bind more substrate -ATCase catalyzes a unique metabolic reaction, alters the rate of catalysis in response to cellular conditions, and responsible for the rate of the larger pathway
Discuss how living organisms regulate the fluidity of their membranes, including in your discussion the effects on fluidity of: temperature, fatty acyl chain length, and number of double bonds.
-longer and/or more saturated FA's are less water soluble -longer and/or more saturated FA's have a higher melting point -The number of double bonds affect the melting point and fluidity because there are many van der Waals packing interactions between saturated chains that are disrupted by the bent cis double bonds of unsaturated fatty acids. -at low temperatures, the fatty acid tails of the phospholipids move less and become more rigid(decreases fluidity, decreases permeability) -at high temperatures, membrane can be damaged or proteins in the membrane can denature
Describe in general terms how cells carry out reversible covalent modification of enzymes, and how the modification would be removed.
-modification of catalytic or other properties of proteins by covalent attachment of a modifying group, cycling between more and less active states, or active and inactive -modification reaction catalyzed by a specific enzyme -modifying group removed by catalytic activity of a different enzyme -covalent modifications generally cause slower and longer-lasting effects than allosteric regulation, maybe coordinated with systemic effects
How does covalent modifications regulate protein activity?
-phosphorylation/dephosphorylation -phosphorylation (phosphoryl transfer from ATP to specific -OH group(s) on protein) catalyzed by protein kinases -dephosphorylation (hydrolytic removal of the phosphate groups) catalyzed by protein phosphatases
Fluid Mosaic Model of Membrane Structure
2-dimensional "fluid" composed of lipids and proteins (both often with attached carbohydrates on outer side of membrane) Fatty acyl chains in the interior of membrane form a fluid, hydrophobic region, with outer surfaces that are hydrophilic Lipids can rapidly diffuse laterally (unless they're anchored to something), but transverse diffusion is very slow in the absence of a catalyst
What is passive transport?
:Spontaneous passage of solute "down" concentration and/or electrical potential gradient, no input of free energy required Simple diffusion(no assistance) Facilitated diffusion(rate enhanced, generally by integral membrane protein); rapid diffusion down a concentration gradient, specific
Briefly explain the results of site-directed mutagenesis experiments with the bacterial serine protease subtilisin, in terms of how much catalytic activity remains (degree of rate enhancement compared to uncatalyzed hydrolysis reaction) after changing the individual members of the catalytic triad to Ala residues, both individual mutations and all three mutations in same enzyme. What general catalytic mechanisms must still be operative in the mutants?
Same catalytic mechanism as mammalian serine proteases: a catalytic Ser assisted by a His and an Asp residue. Mutations in catalytic triad residues have dramatic effects on Kcat(how fast the substrate is produced/turnover number) for subtilisin: transition state stabilization Even when one is removed, it still contributes to catalysis. Mutants must be a very efficient hydrolytic mechanism!
There are four types of proteases based on their chemical mechanisms, all using different methods to make the carbonyl carbon of a peptide bond more susceptible to nucleophilic attack(i.e., more electrophilic), they include:
Serine proteases Cysteine proteases Aspartyl proteases Metalloproteases
Draw the structures of each of the tetrahedral intermediates encountered in the reaction. (If you can do this, you understand the chemistry by which they formed.)
Step 1: When substrate (polypeptide) binds, the side of chain of the residue next to the peptide bond to be cleaved nestles in a hydrophobic pocket on the enzyme, positioning the peptide bond for attack. Histidine extracts one proton from serine to form an alkoxide ion. This serine ion reacts with the substrate. Step 2: In chymotrypsin, the carboxylate R-group of Asp102 forms a hydrogen bond with R group of His 57. When this happens, it compresses this hydrogen bond and shifts electron density to the other nitrogen atom (not involved in the H-bond) in the R-goup of His57 becomes a very strong base. This allows His 57 to deprotonate Ser195 and turn it into a strong nucleophile that can attack the substrate. Oxygen develops a partially negative charge in the oxyanion hole. Step 3: Instability of the negative charge on the substrate carbonyl oxygen when will leads to collapse of the tetrahedral intermediate, re-formation of a double bond with carbon which breaks the peptide bond between the carbon and amino acid group. The amino leaving group is protonated by His57, facilitating its displacement. Once the oxyanion hole stabilizes the negative charge, the bond breaks because the proton from Histidine is binding to nitrogen to make it less likely to carbon. The leaving group is stabilized and the acyl-enzyme is formed. Step 4: The amine component is departed from the enzyme (metabolized by the body) and binds to serine. This completes the first stage (acylation of enzyme). The first product has been made. Step 5: A water molecule is added where the N terminus was. Histidine deprotonates the water to form a hydroxyl group. This hydroxyl group attaches to carbon from the carboxyl side and destabilizes the acyl intermediate. The bond is broken. Step 6: An incoming water molecule is deprotonated by acid-base catalysis, generating a strongly nucleophilic hydroxide ion. Attack of hydroxide on the ester linkage of the acylenzyme generates a second tetrahedral intermediate. Step 7: collapse of the tetrahedral intermediate form the second product, a carboxylate anion, and displace Ser195. The proton from Histidine goes back to Serine. Step 8: The carboxylic acid is released and the enzyme is reformed to catalyze the next reaction with the original active site.
Briefly discuss the structure of calmodulin (± Ca2+), including structure of the "EF hand" motif, and how Ca2+- calmodulin activates target proteins as an example of how a regulatory protein works.
Structure: 4 high-affinity Ca2+ binding sites with "EF hand" structural motif; 2 domains consisting of 2 EF hand motifs and connected by flexible central alpha-helix Target proteins all have a positively charged, amphipathic alpha-helix. Ca2+-CaM binds to positively charged, amphipathic alpha helices in the enzymes, regulating the enzymes activity. CaM kinase I target alpha-helix blocks access of ATP to active site. Binding of Ca2+ calmodulin extracts alpha-helix from CaM kinase I giving ATP to access active site.
What is the role of the enzyme's active site Cys thiol in the glyceraldehyde 3-phosphate dehydrogenase reaction?
The active site of Cys thiol is used for the nucleophilic attack at the aldehyde group of glyceraldehyde 3-Phosphate (G3P) to catalyze its phosphorylation to 1,3-biphosphoglycerate, generating NADH in the process
Explain why amphipathic membrane lipids form self-sealing bilayers in aqueous environments, including the types of interactions stabilizing the bilayer structure.
The bilayer structure of membranes is stabilized by the hydrophobic effect(driving force of bilayer formation), hydration of polar/charged head groups, van der Waals interactions(packing between atoms in hydrophobic core) They form, grow and seal spontaneously because a "hole" would expose the lipid tails to the H2O (which would be entropically unfavorable)
In the chemical mechanism of the hydrolysis of peptide bonds by chymotrypsin, what functional group needs to be made more susceptible to nucleophilic attack by the enzyme?
The catalytic task of a protease is to make that normally unreactive carbonyl(C=O) group more susceptible to nucleophilic attack by H2O.
How does strain(preferential binding of transition state) enhance reaction rate?
The enzyme binds to the transition state very tightly, tighter than its binding to the substrate. Electrostatic interactions help reduce the amount of free energy needed. Energy is needed to break substrate out of the enzyme; if the enzyme binds perfectly to substrate, it doesn't help towards the catalysis of breaking the end
Describe the glyceraldehyde 3-phosphate dehydrogenase reaction, including the overall reaction (substrates and products).
The enzyme takes glyceradehyde-3-phosphatem oxidizes it to a carboxylic acid(3-phosphoglycerate) and transfers the electrons to NAD+, which is reduced to NADH Some of the free energy from the oxidation reaction is conserved by phosphorylating the carboxyl group, generating a mixed anhydride(acyl phosphate). This high-energy bond is able to phosphorylate ADP to ATP in next step of glycolysis(catalyzed by phosphoglycerate kinase)
What does membrane fluidity(essential to membrane function) depend on?
The lipid composition of bilayer. -fatty acid chain length: more C atoms, more packing of tails, higher transition temperature, less fluidity -fatty acid numbers of double bonds(fewer double bonds --> more packing of tails, higher transition temperature, less fluidity) -cholesterol content("buffers" fluidity -- rigid ring structure prevents packing of head groups, but forces packing of double bond "kinked" fatty acid chain
What contributes to the differences in the substrate specificity in serine proteases: trypsin, chymotrypsin, and elastase .
Their structure contributes to the differences in substrate specificity. In chymotrypsin, hydrophobic residues can attach into the pocket. In trypsin, at the bottom of the pocket, it is slightly negative(attracts positive side chain residues) In elastase, there are hydrophobic residues inside, allowing only for the binding of small hydrophobic residues such as Phe, Ala, Gly
What are membrane rafts?
Thickened areas of membrane, stable on a timescale of µsec, enriched in glycosphingolipids and cholesterol, and also enriched with functionally related proteins, e.g., hormone receptor and membrane associated proteins related to signaling function
In transmembrane proteins, what types of amino acid residues tend to cluster at the water-lipid interface, where a transmembrane protein contacts the surface of the membrane?
Tyr and Trp residues tend to cluster in transmembrane proteins at the water-lipid interface, serving as "membrane interface anchors", able to interact simultaneously with the central lipid phase and the aqueous phases on either side of the membrane Positively charged residues (Lys, His, Arg) appear more commonly on the cytoplasmic face of membranes.
What are channels in passive transport?
Very rapid, i.e., ion chnnale: ~10^7-10^8 ions/sec, "down" a concentration gradient not saturable, degree of specificity/ion selectivity varies gated ion channels(ligand- or voltage-gated = control)
For uncharged solutes the free energy of transporting across membrane depends only on the chemical concentration gradient across the membrane; what equation shows this?
dGt = RTln(c2/c1) at equilibrium: dGt = 0 when c1=c2 frr an uncharged solute
Know the definitions of: : first committed step, multimeric protein, feedback inhibition, cooperativity, cooperative binding, T state, R state, allosteric, homotropic effector/modulator/regulator, heterotropic effector/modulator/regulator, allosteric activator (positive hetero-/homo-tropic effector/modulator/regulator), allosteric inhibitor (negative heterotropic effector/modulator/regulator), isozyme, "EF hand" motif, target enzyme, kinase, protein kinase, phosphatase, protein phosphatase, target protein, cAMP, consensus sequence, pseudosubstrate, activation loop and P loop (in protein kinase structures and regulation), cascade, reciprocal regulation, zymogen
definitions
Know the definitions of: micelle, lipid bilayer, amphipathic, ganglioside
definitions
Integral Membrane Proteins
interact with hydrophobic core of bilayer lipids, and are removable only by detergent treatment, organic solvents, etc. that disrupt the bilayer
Discuss the concepts of lateral and transverse ("flip-flop") diffusion of membrane lipids and proteins, and the asymmetric distribution of membrane components (especially carbohydrate portions) on the extracellular and intracellular sides of the bilayer.
lateral diffusion: rapid for both proteins and lipids within the plane of the membrane(except for anchored proteins) transverse diffusion: ("flip-flop") of both proteins and lipids is extremely slow, unless mediated by protein "flippases" Lipid composition in the 2 leaflets of bilayer is asymmetric, as is protein distribution. Fatty acyl chains in the interior of the membrane form a fluid, hydrophobic region, with outer surfaces that are hydrophilic. Lipids can rapidly diffuse laterally (unless they're "anchored" to something),but transverse diffusion is very slow in the absence of a catalyst. The inner and outer monolayers (or "leaflets") of the membrane bilayer have different lipid compositions. Integral membrane proteins always have a specific orientation, with specific regions pointing into the cytoplasm and others pointing out to the cell exterior. The carbohydrate components on glycolipids and glycoproteins are almost always found on the outer surface of the membrane. This asymmetry is maintained because of the extremely slow rate of rotation of components across the membrane. The overall lipid composition of a given membrane/leaflet is related to the cell's environment (especially temperature).Lipid composition regulates fluidity.
What are the three membrane lipids(amphipathic)?
membrane lipids are responsible for spontaneous formation of lipid bilayers -glycerophospholipids: glycerol backbone 2 fatty acyl tails in ester linkage + a polar head group(a phosphate ester of another alcohol like choline, ethanolamine, serine, inositol, etc.) -sphingolipids: sphingosine backbone (1 tail) + fatty acid chain in amid linkage (another "tail") + either carbohydrate (glycosidic bond to sphingosine) or phosphate ester of another alcohol like choline or ethanolamine(ester bond to sphingosine): includes glycosphingolipids, phosphosphingolipids -cholesterol
Prostaglandin H2 Synthase(membrane protein)
on surface of ER membrane but anchored in membrane by a set of alpha-helices with hydrophobic R groups that extend into membrane core
Terminology: peripheral, integral, amphitropic, lipid-linked, transmembrane helix; antiparallel beta-barrel; hydropathy plot
operational definition - how it works
What does catalysis involving a functional group whose state of ionization affects the catalytic activity(either an enzyme or a substrate) depend on?
pH
Glycophorin(membrane protein)
single hydrophobic transmembrane alpha-helix
What is active transport?
transport of solute against its concentration gradient requires exergonic process to drive uphill transport, primary active transport(transport of solute against its concentration gradient, coupled directly to an exergonic chemical reaction Secondary active transport(transport/flow of one solute down its concentration gradient is used to drive transport of a different solute against its concentration gradient)
What are transporters in passive transport?
works by conformational changes linked to ligand binding, rapid transport "down" concentration gradient, saturable(shows a maximum velocity; can measure Kt analogous to Km), specific
How does proteolytic activation(zymogen activation) regulate protein activity?
• digestive proteases like chymotrypsin and trypsin • blood clotting cascade
