biochem quiz 3

proteolytic cleavage for viral activation

• SARS-CoV-2 translates its genome into large, inactive polyproteins (PP1a and PP1b)

• polyproteins must be cleaved at specific recognition sites by the main protease (M^pro / Nsp5) to produce functional viral enzymes

active site target for enzyme inhibition

• active site of M^pro contains a catalytic cysteine residue (Cys145) within its substrate-binding pocket

• this makes it a prime therapeutic target for covalent, competitive enzyme inhibitors

enxyme taregt and pathway control

• atorvasatin targets HMG-CoA reductase, the enzyme that catalyzes the rate-limiting step of cholesterol biosynthesis → controls lipid production

competitive inhibition mechanism

• atorvastatin acts as competitive inhibitor by structurally mimicking the natural substrate HMG-CoA

• the HMG-like side binds directly to the enzyme’s active site, blocking the real substrate from entering and stopping the pathway

first-order rate law

• the overall rate of reactant disappearance or product formation depends directly on a single reactant concentration

• slope = -k

v = d[P] / dt = -d[A] / dt = k[A]

seocnd order rate law

• when a reaction involves the collision/interaction of two molecules, the reaction order matches the molecularity

• self-collision (2A → P): rate depends on the square of reactant A

  • v = -d[A]/dt = k[A]²

• two different species (A + B → P): rate depends on both reactants

  • v = -d[A]/dt = -d[B]/dt = k[A][B]

michaelis-menten kinetics

• v₀ = v_max[S] / K_m + [S]

• v_max = k₂ [E]_T

• describes how initial reaction velocity depends on substrate concentration

• at low substrate concentrations the rate is first-order with respect to substrate

• at high substrate concentrations the enzyme is fully saturated and the reactions shift to zero-order

lineweaver-burk equation

• double-reciprocal plot transforms the hyperbolic michaelis-meten equation into a straight-line equation

• slope = K_M / v_max

• y-intercept = 1 / v_max

• x-intercept = -1 / K_M

1/v₀ = (K_M / v_max) 1/[S] + 1/v_max

enzyme reaction intermediates

• enzyme-catalyzed reactions frequently proceed through multiple enzyme-bound intermediates from the initial enzyme substrate complex to the enzyme-product complex

“black box" limitation of steady-state kinetics

• only measures the input and the output so the mechanism is an unobservable “bllack box”

• due to this, the steady-state kinetic data can’t reveal the specific sequence of intermediates inside

bisubstrate reaction

• involves two substrates reacting together to form two products catalyzed by an enzyme

• require specific binding mechanisms to handle multiple substrate in a single enzyme

group transfer mechanism

• many bisubstrate reactions function as a group-transfer processes where a chemical group is transferred from one molecule to another

• the enzyme facilitates the movement of the functional group between the donor and acceptor molecules

Hydrolysis/Trypsin

• trypsin catalyzes a bisubstrate reaction involving a polypeptide and water to cleave peptide bonds

• create carboxylate and amine products

Oxidation/Alcohol Dehydrogenase

• alcohol dehydrogenase catalyzes group/atom transfer between an alcohol and NAD⁺ to form a ketone/aldehyde and NADH

ordered bisubstrate reaction

• strict binding sequence means substrate A must bind to the free enzyme first to form complex before substrate B can bind

• products are also released a specific sequence where product P departs first followed by Q before returning the free enzyme

random bisubstrate reaction

• flexible binding order → either subsrtate A or B can bind to the free enzyme to create the complex

• both binding routes lead to the same central quaternary complex from which products can be released in random order

ping pong bisubstrate reaction (double-displacement)

• first substarte binds and transfer a chemical group to the enzyme, releasing the first product and leaving behind a modified enzyme form

• when B binds to form the modified complex and reacts to release the seocnd product for a ternary complex of all reactant is never formed at the same time

succinate dehydrogenase and inhibition

• succinate dehydrogenase catalyzes the oxidation of succinate to fumarate

• malonate acts as a competitive inhibitor (bc its structure is similar to succinate) which allows it to bind to the active site

• but it cannot undergo the reaction so no reaction

competitive enzyme inhibition

• competitive inhibitor directly competes with substrate by reversibly binding to free enzyme with a dissociation constant K_I to form an enzyme-inhibitor complex

• since the active site is blocked adding substrate to the complex produced no reactions

ethanol treatment of methanol posioning

• alcohol dehydrogenase processes both methanol and ethanol, methanol oxidizes into toxic formaldehyde while ethanol oxidizes into acetaldehyde

• ethanol acts a competitive inhibitor agaisnt methanol by outcompeting it for the enzyme’s active site

competitive enzyme inhibition michaelis-menten kinetics

• as inhibitor concentration increases, the K_m increases meaning a higher substrate concentration is required to reach half-max velocity

• at high substrate concentrations, the substrate outcompetes the inhibitor which allows the reaction velocity to reach the true v_max

competitive enzyme inhibition (lineweaver-burk plot)

• all lines with inhibitor concentratiosn intersect at the exact same y-intercept (1/v_max)

• as inhibitor concentration increases the slope increases and the x-intercept shift closer to zero

uncompetitive enzyme inhibition

• the inhibitor does not bind to the free enzyme but binds exclusively to the already-formed enzyme-substrate complex to create an inactive enzyme-substrate-inhibitor complex

• yields no reaction and cannot breakdown into product which locks the substrate in place

uncompetitive enzyme inhibition lineweaver-burk plot

• bc both the apparent v_max and K_m decrease by exact same factor, the lineweaver-burk plot lines for increasing inhibitor concentrations remain perfectly parallel with constant slope

• as inhibitor increases, the y-intercept shift upward and the x-intercept shift to the left

noncompetitive enzyme inhibition mechanism

• inhibitor can reversibly bind to either the free enzyme with a dissociation constant K_I or the enzyme-substrate complex

• both resulting pathways lead to no reaction and cannot yield product

noncompetitive enzyme inhibition lineweaver-burk plot

• bc the inhibitor binds to E and ES with unequal affinities both the slope and the y-intercept change as inhibitor concentration increases

major glands of endocrine system

• hormones help the body maintain homeostasis, respond to external stimuli and manage developmental cycles

• key glands: hypothalamus, pituitary, thyroid, parathyroid, adrenal glands, pancreas, kidneys, ovaries and testes

endocrine signaling

• specialized endocrine cells synthesize hormone molecules and secrete them directly into the circulatory system for transport

• hormones circulate through the bloodstream until they encounter target cells equipped with specific matching receptors, allowing them to bind and elicit a cellular response

adrenal medulla synthesizes two hormonally active catecholamines

• both hormones share catecholamine backbone consisting of a benzene ring with 2 OH group to an ethylamine side chain containing a beta-hydroxyl group

• R group atatched to the terminal nitrogen: R = H forms norepinephrine and R = CH₃ forms epinephrine

synthesis of catecholamines

• amino acid tyrosine is converted into L-DOPA via tyrosine hydroxylase followed by aromatic amino acid decarboxylase removing CO₂ to yield dopamine

• dopamine beta-hydroxylase converts dopamine into norepinephrine, and phenylethanolamine N-methyltransferase uses S-adenosylmethionine to add a methyl group, completing the transformation into epinephrine

glucocorticoids

• influence carbohydrate, protein and lipid metabolism in ways nearly opposite to insulin, while also managing inflammatory reactions and coping with stress

mineralocorticoids

• primarily regulate salt and water excretion in the kidneys, whereas androgens and estrogne direct sexual development and reproductive function

growth hormone receptor dimerizes upon hormone binding

• the binding of a growth hormone molecule induces a structural conformational change that forces two separate receptor monomers to associate and form a functional dimer complex

• this hormone-induced dimerization brings the intercellular domains close together near the inner part of the plasma membrane to kick off downstream kinase signaling

metabolic regulation

• every signaling pathway consists of a receptor, a mehanism for transmitting the ligand-binding event inward and intracellular cascades involving second messengers or kinase/phosphatase modifications

• Ras cascade, adenylate cyclase generating cAMP and phospholipase C

specific g-protein coupling

• primary g-proteins that activates adenylate cyclase is G_s whereas the major g protein linked to phospholipase c activation is G_q

• biological response depends on receptor availability, concentration if intracellular components, and how the cell integrates simultaneous signals from multiple competing ligands

reaction of protein tyrosine kinases

• protein tyrosine kinases transfer a phosphate group from ATP to the hydroxyl group located on the aromatic ring of a target tyrosine residue within a protein

• yields a phosphorylated tyrosine side chain along with ADP as a byproduct

autophosphorylation activates insulin receptor

• binding of the hormone triggers structural rearrangements and autophosphorylation across the receptor subunits to shift the protein into an active signaling state

tyrosine kinase domain of insulin receptor

• active pocket accommodates nucleotide substrates and cofactors, enabling the precise transfer of phosphate groups to target amino acid residues

conformational change on phosphorylation

• phosphorylation induces a significant physical rotation and backbone rearrangement within the kinase domain

• induced conformational rotation opens or closes active loops, shifting the enzyme between inactive and fully active catalytic conformations

SH2 domains bind phospho-tyr residues

• SH2 domain specifically recognizes and binds phsopotyrosine (pTyr) residues along with the downstream adjacent amino acid sequence

• by binding selectively to phosphorylated tyrosine motifs, SH2 domains act as molecular adaptors that recruit specific downstream target proteins to activated receptor complexes

ras signaling cascade

• binding a growth factor to a RTK triggers recpetor autophsophorylation creating phosphotyrosine sites that recruit the adaptor via its SH2 domain which the guanine nucleotide exchange factor Sos to activate Ras by replacing GDP with GTP

dowstream kinase ascade and gene expression

• activated Ras GTP triggers a sequential phosphorylation cascade through Raf → MEK → MAPK, activated MAPK then translocates into the nucelus to phosphorylate transcription factors

Ras Signaling Cascade – Developmental Disease

• mutations affecting components of the Ras-MAPK signaling pathway result in various developmental disorders, including Noonan syndrome, Costello syndrome, LEOPARD syndrome, and neurofibromatosis type 1

• conditions often stem from dysregulated positive or negative feedback loops that perturb normal cellular growth and differentiation during development

Ras Signaling Cascade – Cancer

• mutations that hyperactivate the Ras pathway involve gain-of-function alterations in receptor tyrosine kinases, GEFs, or Ras isoforms as well as downstream effectors like RAFs and MEKs

• loss-of-function mutations in negative regulators, such as GAPs , Sprouty, or CBL remove crucial inhibitory brakes on the cascade, driving unchecked cellular proliferation, cyclin expression, and S-phase entry

SH3 domains bind pro-rich polypeptides

• SH3 domains specifically recognize and bind to proline-rich peptide motifs located on partner proteins

• binding surfaces mediate critical protein-protein interactions that assemble modular signaling complexes and localize proteins near cell membranes

SH2 & SH3 domains of Grb2 indirectly activate Ras

• adaptor protein Grb2 contains a central SH2 domain flanked by two SH3 domains allowing it to bridge receptors and downstream effectors

• SH2 domain binds to phosphorylated tyrosine residues on activated receptors, while the SH3 domains recruit proline-rich guanine nucleotide exchange factors (like Sos) to activate Ras

RasGAP Activates the GTPase Activity of Ras

• RasGAP complex positions a "finger loop" containing an essential arginine residue R789 into the active site of Ras to neutralize charges and stimulate its intrinsic GTPase activity

MAP kinase cascades

• function through a conserved three-tiered module consisting of a MAP kinase kinase kinase which phosphorylates and activates a MAP kinase kinase, which in turn activates the terminal MAP kinase

• different upstream stimuli activate distinct pathway branches involving specific kinases to regulate targeted transcription factors and drive cellular growth, differentiation, survival, or apoptosis

Src structure reveals mechanism of autoinhibition

• 3D structure of c-Src shows that the regulatory SH2 and SH3 domains fold back to interact directly with the protein tyrosine kinase core domain

• internal clamping arrangement locks the kinase into a closed, autoinhibited state by blocking access to the active site and critical phosphorylation sites like Tyr416

Src Activation

• Src activation occurs when an external activator protein binds via a proline-rich motif to the SH3 domain, and phosphorylated tyrosine residues bind to the SH2 domain

• competitive displacement releases the intramolecular clamp, freeing the kinase domain and allowing autophosphorylation at Tyr416 to fully activate the enzyme for downstream substrate phosphorylation

PTKs as Anticancer Targets

• PTKs frequently become dysregulated or mutated in cancers, making them prime targets for small-molecule drug inhibitors

• anticancer drugs are designed to bind tightly within the conserved ATP-binding catalytic cleft of the kinase domain, which blocks phosphate transfer and halts oncogenic signaling pathways

Protein Tyrosine Phosphatase SHP-2

• contains N-terminal and C-terminal Src homology domains that act as a regulatory switch controlling access to the catalytic protein tyrosine phosphatase domain

• the catalytic pocket contains the conserved CX₅R signature motif which uses specific cysteine and arginine residues to execute the nucleophilic attack required to remove phosphate groups from phosphotyrosine residues

protein phosphatase PP2A

• contains an extensive horseshoe-like scaffolding structure built from tandem strucutral repeats

• the scaffolding subunit coordinates the assembly of regulatory and catalytic subunits into a functional multimeric holoenzyme complex capable of dephosphorylating diverse intracellular target proteins

Adenylate Cyclase Reaction

• adenylate cyclase catalyzes the conversion of ATP into cAMP by forming a cyclic bond between the 3’ hydroxyl group and the alpha phosphate

cAMP-Bound Bovine Protein Kinase A Regulatory Subunit

• binding of cAMP triggers a conformational change that causes the regulatory subunits to dissociate which releases active catalytic subunits to phosphorylate target proteins

PKA Activation Cascade

• adenylate cyclase converts ATP into cAMP which binds to the regulatory subunits of protein kinase A which releases active catalytic subunits to drive downstream cellular responses

Modulators That Affect Cell Signaling

• Caffeine, theophylline, and theobromine share a conserved xanthine ring structure substituted with methyl groups at specific ring positions

• act as key cellular modulators that prolong cAMP signaling duration and intensity

Drugs & Toxins That Affect Cell Signaling

• uses feedback loops to coordinate amplification

• pathogenic factors covalently modify specific g-protein alpha subunits which disrupt normal GTPase activity and locking the signaling pathway

Phosphoinositide Signaling System

• ligand binding triggers g-protein coupled receptor to activate gtp complex which in turn stimulates phospholipase C to cleave membrane-bound PIP₂

• this cleavage produces IP₃ which triggers Ca²⁺ release from the ER and DAG which activates protein kinase to drive downstream cellular responses

Phospholipase C Reaction Yields 2nd Messenger IP₃

• phospholipase C uses water to cleave PIP₂ into DAG and IP₃

• while DAG remains anchored in the plasma membrane, IP₃ is releases as a soluble cytosolic secondary messenger to stimulate calcium channels

Calmodulin: Ca²⁺ Activated Switch

• calmodulin contains flexible central linker domains flanked by globular lobes capable of binding calcium ions

• binding of Ca²⁺ induces a major conformational chnage that allows calmodulin to bind and regulate enzymes

EF Hand

• motif consists of a helix-loop-helix structural arrangement

• specific amino acids within the loop region provide oxygen ligands that securely binds the Ca²⁺ ion

Ca²⁺-CaM-dependent Protein Kinase Activation

• in an inactive state protein kinase’s regulatory domain blocks its active site

• once activated, the kinase binds substarte proteins, uses ATP to phosphorylate them and triggers downstream cellular responses

Protein Kinase C Activated by Diacylglycerol

• C1 domain of protein kinase C contains zinc ions that stabalize a binding cleft specifically tailored for DAG

• binding of DAG to this domain increases the hydrophobic surface area of PKC which drives its translocation to the plasma membrane where it becomes fully active to phosphorylate downstream targets

Insulin Signaling in Liver

Decreases glucose production and increases glucose utilization

Insulin Signaling in Muscle

Increases glucose uptake

Insulin Signaling in Fat

Increases glucose uptake and triglyceride synthesis while decreasing lipolysis

Temporal Regulation in Seconds

Involves rapid events including PIP3 production

Temporal Regulation in Minutes

Involves intermediate steps regulating TSC2

Temporal Regulation in Hours

Involves long-term transcriptional control through SREBP1c

Chemogenetics

A drug-based approach for controlling cell function that features poor temporal control

Optogenetics

A light-based approach providing spatiotemporal control but requiring implants due to poor tissue penetration

Magnetogenetics

An approach using radio waves and magnetic nanoparticles like ferritin with TRPV1 channels to achieve spatiotemporal control with free tissue penetration

Bacteriorhodopsin

A light-driven proton pump that transports protons out of the cytoplasm

Channelrhodopsin

A light-gated cation channel permeable to sodium

Halorhodopsin

A light-driven chloride pump that transports chloride ions into the cytoplas