biochem exam 3 written study (enzyme mechanism, chymotrypsin, lipids)

what is a nucleophile?

-nucleophiles have excess electrons

-generally more nucleophilic if negative charge and that negative charge isn’t delocalized

-less electronegative, smaller atoms are more nucleophilic

-serine and threonine (O-), cysteine (S-), lysine (uncharged amine), histidine (imidazole), water (OH-)

what is an electrophile?

-electron deficient

-positively charged or double bounds (polar)

-carbon of carbonyl (C=O), protonated imine group (C=N), phosphorous of phosphate group

-usually substrates, not the enzymes

what is chymotrypsin?

-chymotrypsin is a protease: catalyzes hydrolytic cleavage of amide (peptide) bonds

-digestive enzyme found in pancreatic juice

how does chymotrypsin work?

-stabilizes transition state

-general acid-base catalysis and covalent catalysis (enzyme covalently attached to substrate)

what is the chymotrypsin active site?

-hydrophobic pocket called S1 and the residues required for catalysis

chymotrypsin is a serine protease (uses serine), what is chymotrypsin’s catalytic triad?

-serine, histidine, and aspartic acid in a hydrogen bonded network

proteases often cleave peptide backbone next to an AA they are designed to interact with. where does chymotrypsin cleave bonds?

-chymotrypsin cleaves peptide bonds next to large hydrophobic residues

-the S1 pocket is large and hydrophobic

what does the H bonding network of histidine help do?

-the H bonding network of histidine helps make serine more acidic, that proton wants to come off more

what is the role of the aspartic acid in the catalytic triad?

-asp helps anchor histidine in the correct position, stabilizes the build up of positive charge on histidine

-favors tautomer of neutral histidine with a proton on the delta 1 nitrogen, not delta 2 because that wouldn’t increase nucleophilicity of serine

-lone pair is on the epsilon nitrogen closer to serine, used to deprotonate serine

what is the substrate for chymotrypsin?

-chymotrypsin’s substrate is a polypeptide

-not reading N→C, reading C→N

what is an oxyanion hole?

-oxyanion hole: a pocket in active site that stabilizes transition state tetrahedral intermediate

-tetrahedral intermediate has (-) charge on deprotonated oxygen

-pocket consists of backbones amides or positively charged residues

what occurs in the non-enzymatic peptide hydrolysis mechanism?

-water is used to break the peptide bond

what are the first steps of non-enzymatic peptide hydrolysis?

-water attacks peptide bond carbonyl carbon in basic conditions

-then you get a tetrahedral intermediate with OH2+

-then you need to deprotonate that OH2+ to destabilize it, and protonate the HN peptide part to make that a good leaving group

what are the later steps of non-enzymatic peptide hydrolysis?

-a proton transfer occurs to deprotonate OH2+ → OH and protonate NH → NH2+

-this is not particularly likely unless in acidic conditions

-the O- of the tetrahedral intermediate attacks and the tetrahedral intermediate collapses, the NH2+ part of the peptide leaves

-the peptide bond is now broken

what does chymotrypsin do, why do you need it?

-this reaction is really hard to do without an enzyme, need particular acid/base conditions

-chymotrypsin makes the initial nucleophile more nucleophilic (serine pka decreased)

-chymotrypsin stabilizes the tetrahedral intermediates with H bonding

what is the first step of chymotrypsin’s mechanism?

-histidine grabs an H from serine

-serine’s oxygen electrons (nucleophile) attack the carbonyl of the peptide bond, breaking the carbonyl double bond and forming a tetrahedral intermediate

what is the second step of chymotrypsin’s mechanism?

-the unstable tetrahedral intermediate collapses: the electrons on the oxygen come down to attack the bond (reforming pi bond)

-then the bond actually attacks the histidine hydrogen, the NH bond is grabbing a proton from the histidine, making it a good LG

-the bond is broken, no proton transfers needed like the non-enzymatic mechanism

what is the third step of chymotrypsin’s mechanism?

-now we have an acyl enzyme intermediate (C=O attached to serine O) and new n terminus

-a water molecule is deprotonated by base catalysis, forming an OH- (histidine takes the proton from water)

-the OH- attacks the ester linkage of the acyl-enzyme to make another tetrahedral intermediate

-the oxygen in the oxyanion hole has a negative charge now and histidine is positively charged

what is the fourth step of chymotrypsin’s mechanism?

-the tetrahedral intermediate collapses to form a carboxylate anion which then displaces serine

-to displace serine it is similar to previous steps, electrons from the oxygen are pushed around to kick off the bond attached to the O of serine

-and those electrons from the serine bond grab a proton from the protonated histidine

-left with peptide product 2 broken off, new c terminus

what does chymotrypsin look like after the mechanism?

-exactly the same, the enzyme should always be regenerated

is the chymotrypsin reaction energetically favored?

-yes this reaction is energetically favored

-exothermic, spontaneous, exergonic so -delta G and -delta H

-no energy used

what is the buildup of negative charge on the carbonyl of the tetrahedral intermediates of the chymotrypsin stabilized by?

-the buildup of negative charge on the carbonyl is stabilized by H bonding with the enzyme backbone and oxyanion hole

what is the E+S reaction for the substrate binding? how does this look for when there is the intermediate of the covalently attached peptide to the ES complex?

-E + S → ES represents the chymotrypsin enzyme binding to its peptide substrate

-E + S → ES → ES* represents going from the chymotrypsin-peptide complex to now chymotrypsin’s serine being covalently attached to the ES complex

what is the reaction of E+S going to the first product, ½ of the polypeptide cleaved?

E+S → ES → ES* → ES* → P1

binding → serine attack → collapse of tetrahedral intermediate → 1st product

what is the full E+S reaction for chymotrypsin?

E+S → ES → ES* → ES* → P1 → ES* + H2O → ES** → EP2 → E + P2

-where now after product 1, water comes in to form the second covalently bound ES complex which then will turn into the enzyme + product and lastly the enzyme is cleaved

-these aren’t always irreversible arrows also

does chymotrypsin cleave on the N or C terminal side of a hydrophobic residue?

-chymotrypsin cleaves on the C terminal side of a hydrophobic residue, it will be closest to R1

-the order goes N term, R3, R2, R1, C term

who does chymotrypsin utilize general acid-base catalysis?

-base: histidine deprotonates serine and deprotonates water

-acid: histidine protonates the NH leaving group and protonates serine

how does chymotrypsin utilize covalent catalysis?

-serine reacts with the NH to form an acyl enyme intermediate, serine is covalently attached to the peptide

how does chymotrypsin use transition state stabilization?

-the oxyanion hole stabilizes the high energy tetrahedral intermediate through H bonding

what is HIV? what is reverse transcriptase and integrase?

-HIV is a retrovirus (RNA genome)

-reverse transcriptase synthesizes a DNA copy, so going RNA → DNA

-integrase inserts the now genomic DNA into the host genome

what cleaves the large HIV viral polyproteins?

-HIV protease, an aspartic protease

-the proteins are polyproteins because it is harder to hijack a host ribosome multiple times for many proteins

what does HIV protease favor having?

-proline

-so if you have Phe-Pro, after it is cleaved you end with Phe-COOH and N-term-Pro

what is the mechanism for HIV protease?

-aspartic acid increases the nucleophilicity of water by taking a proton from it

-the now OH- attacks the carbonyl

-a tetrahedral intermediate forms that is stabilized by the OH on another aspartic acid (unlike chymotrypsin this isn’t covalently attached to the enzyme)

-the tetrahedral intermediate collapses and the bond attached to the proline will grab a proton from the aspartic acid with an OH to make it a good leaving group

what are the HIV protease inhibitors?

-transition state analogs

-bind active site reversibly → do not react

what is the difference between the transition state of the HIV protease inhibitors and the transition state seen without the inhibitor?

-the inhibitor doesn’t have a carbonyl, it has an OH, so there can’t be any attack on an electrophilic center to begin with

-this analog can H bond with the enzyme better than the actual substrate

what are the two step proteases? what are the two steps?

-serine proteases with the catalytic triad

-cysteine proteases with a catalytic dyad

1. covalent intermediate

2. activated water to hydrolyze acyl enzyme intermediate

*just bc it is called 2 step doesn’t mean there are only 2 steps

what are the one step proteases? what is the one step?

-aspartic acid proteases

-metallo-protease: metal complex

1. activated H2O hydrolyzes the peptide bond

what exists in metallo-proteases, what are they doing?

-glutamate increases nucleophilicity of H2O

-zinc (lewis acid) complexed with histidine also increases nucleophilicity of H2O and coordinates substrate

trypsin is a serine protease that cleaves on the C-terminal side of lysine and arginine residues. what do you expect is different between trypsin and chymotrypsin?

-chymotrypsin cleaves near nonpolar aromatics (Phe, trp.) so it has a nonpolar hydorphobic S1 pocket

-trypsin cleaves near positively charged residues (arg, lys) so it probably has negative amino acids in its pocket that can salt bridge with these

-basically the active site is different between the two because they have different substrates

cysteine proteases are similar to serine proteases, except the serine is replaced by cysteine. cysteine proteases don’t need Asp as much, they may only have a catalytic dyad of His and Cys. why is this possible for cys proteases but not ser?

-cysteine has a sulfur instead of an oxygen

-sulfur is less electronegative so it is more nucleophilic

-more nucleophilic means it doesn’t need the asp to increase nucleophilicity

-cys pka = 8, ser pka = 15 so cys is probably halfway deprotonated anyway

how are enzymes regulated?

-can be constitutively expressed/active

-or can be only expressed in response to certain signals

what is the point of having regulation of enzymes?

-allows a cell to meet changing needs for energy and biomolecules

-why waste resources on something that isn’t needed?

how is enzyme activity controlled?

-enzyme activity is primarily controlled through regulation of [E] + catalytic activity

what does aspartate transcarbamoylase (ATCase) do?

-ATCase catalyzes an early step in the biosynthesis of pyrimidine nucleotides (DNA, RNA)

what happens to ATCase when substrate binds?

-when substrate binds to ATCase T state, the protein’s structure gradually transitions from the inactive T state to the active R state

-6 catalytic units and 6 regulatory units

-sigmoidal kinetics because of this allostery

do allosteric enzymes exhibit michaelis-menten kinetics?

-no, allosteric enzymes are sigmoidal instead of hyperbolic

-this means no Km, the equivalent would be [S]0.5 or K0.5

how do small changes in [S] change activity?

-small changes in [S] can result in large changes in activity, the steep part of the sigmoidal curve

-being off, turning on this is almost like a step function and is useful for an enzyme you don’t want always working

ATCase is heterotropically modulated by what modulators?

-ATP and CTP

what kind of modulator is ATP on ATCase?

-ATP is a positive modulator (stimulant/activator)

-high ATP indicates the cell is growing, so ATCase needs to be active (R state)

-positive feedback mechanism

what kind of modulator is CTP on ATCase?

-CTP is a product of the pathway, high CTP indicates ATCase is not needed

-CTP binds as a negative/inhibitory regulator (favors T state)

-negative feedback loop

what is the effect of positive modulators on K0.5 and Vmax?

-positive modulators decrease K0.5 and don’t change Vmax

-more hyperbolic

-increased rate at the same [S]

what is the effect of negative modulators on K0.5 and Vmax?

-negative modulators decrease K0.5 and don’t change Vmax

-decreased rate at the same [S]

*but this isn’t an inhibitor

can modulators change Vmax?

-yes, some modulators alter Vmax without changing K0.5 much

-these modulators either facilitate or slow catalysis without altering binding affinity

-these are less common

what is regulation by reversible covalent modification?

-some AAS are susceptible to covalent modification

-some enzymes specialize in the addition or removal of covalent modifications

-think epigenetics: phosphorylation, ubiquitination, acetylation, methylation

what does covalent modification do to a protein?

-covalent modification can activate/inactivate an enzyme

-can induce change in conformation

-can cause associations between protein and other biomolecules

what is phosphorylation?

-adding phosphate to an enzyme

-you need an oxygen based nucleophile for this (try, ser, thr) or histidine’s imidazole N

-phosphorylation introduces a negative charge that can cause changes in protein conformation, substrate binding, catalytic activity

how do kinases work? how do phosphatases work?

-kinases: use ATP to covalently attach a phosphate group to other proteins

-phosphatases: remove phosphoryl groups

what is the example we discussed of phosphorylation causing dramatic structural rearrangement?

-PINK1 enzyme phosphorylates ubiquitin ser, which enhances binding affinity for parkin

-mitochondria protein kinase PINK1 phosphorylates parkin’s ser residue in the UBL domain

some proteins are expressed in an inactive form, what are these called? what happens when these need to be used?

-zymogen, proenzyme, proprotein

-zymogen is irreversibly cleaved to form active enzyme

-both chymotrypsin and trypsin are regulated this way

what is happening with the inactive form of chymotrypsin, chymotrypsinogen?

-new N terminus forms ionic bond with Asp, triggering conformational changes

-Met moves to surface, the hydrophobic pocket is created upon cleavage

-NH groups that stabilize oxyanion move into place

what are biological lipids?

-amphipathic molecules → polar + nonpolar end

-fats and oils are used for membrane formation

-structural elements of plasma membrane

-enzyme cofactors

-hydrophobic anchors for proteins

-hormones and signaling molecules

what are fatty acids?

-fats and oils used almost universally for energy storage are derived from fatty acids

-carboxylic acid with long alkyl chain

what is a saturated fatty acid? what is an unsaturated fatty acid?

-saturated= all single bonds (saturated with hydrogens)

-unsaturated= double bonds, usually cis configuration

what are transfats? what are omega-3-w-3 fatty acids?

-transfats: product of fermentation of dairy animals (milk-based and meat)

-omega-3-w-3 fatty acids have a double bond between C3 and C4

how does length and double bonds impact solubility?

-more length= less soluble

-fewer double bonds = less soluble

are unsaturated or saturated fatty acids easier to melt?

-unsaturated fatty acids are easier to melt because they have kinks in packing, they aren’t packed as closely

what are triacylglycerols (triglycerides)?

-composed of glycerol attached to fatty acids with an ester linkage

-can be homo or heterogenous

-nonpolar and extremely hydrophobic (not amphipathic, no charge because of ester)

-fuel storage in animals and plants

what are lipases?

-enzymes that cleave ester linkages

what are glycerophospholipids (phosphoglycerides)?

-long alkyl chain with kink due to cis double bond

-hydrophilic head with glycerol, phosphate, and choline head

where are sphingolipids and what are they involved in?

-sphingolipids are typically found in neurons

-in multiple sclerosis the immune system attacks and destroys sphingosines

-like glycerolipid but instead of glycerol there is an amide linkage. glycerolipid could do carbonyl chemistry or aldol stuff

what are sterols like cholesterol?

-sterols are structural lipids

-steroid nucleus is common structure: 4 fused rings

-no rotation around C-C bonds

-amphipathic (polar head group OH)

-membrane components

-steroid hormones

what are lipid nanoparticles used for?

-mRNA vaccine delivery

-thousands of lipids encapsulate mRNA to shield from enzymes and deliver to cells via membrane fusion

what lipid nanoparticles were used in COVID-19 vaccines?

-ionizable lipids: positive charges bind negative RNA backbone

-pegylated lipids: stabilize/solubilize particle

-phospholipids and cholesterol: contribute to particle structure

what are integral membrane proteins? what is monotopic vs. polytopic?

-integral membrane proteins are embedded in lipid bilayer

-monotopic: interacts with 1 leaflet of layer (half of the layer)

-polytopic: protein traverses full membrane 1 or more times (can be critical in signaling bc they know what is happening inside and outside of cell)

what are amphitropic proteins?

-amphitropic proteins associate reversibly with membranes, mostly interacting with surface of membrane

-found in membrane and cytosol

-association often regulated by phosphorylation or ligand binding causing conformational change

what are the properties of protein with single transmembrane domain?

-N term and C term domains are hydrophilic → inside/outside cell

-transmembrane domain mainly hydrophobic

-specific orientation in bilayer, same side always outside/inside

-glycosylated residues always on extracellular face of bilayer

what do hydropathy plots do?

-hydropathy plots predict transmembrane regions

-to predict how many transmembrane domains look at number of peaks that dip down