BIOL 4087 Lee - Exam II

Drug Design: Diagnostic 2-DG

2-deoxyglucose (2-DG) is a synthetic glucose analog; 2-hydroxyl group replaced with H 2-DG inhibits hexokinase II (which catalyzes the initial metabolic step in conversion of glucose) and suppresses glycolysis Due to the Warburg effect, cancer cells consume more glucose than normal cells. 2-DG analogs are used to detect transformed cells vis PET scans (cells transformed to tumor cells as a consequence of their enhanced glucose uptake and dependence on glycolysis)

Active Sites

3D structures where substrates bind to enzymes through multiple non covalent interactions They may assume a new conformation after the substrate binds (induced fit model)

redox reaction

A chemical reaction involving the transfer of one or more electrons from one reactant to another Also called oxidation-reduction reaction

Transition state analog

A stable molecule that resembles the transition state of a particular reaction, and therefore binds the enzyme that catalyzes the reaction more tightly than does the substrate in the ES complex

Non-Vitamin sourced coenzymes

ATP SAM UDP

Coenzyme: Vitamin C

Absorbic acid ROS fighter Reducing reagent (ROS-reducing agent working together with Vitamin E) (anti-oxidant coupled with vitamin E) Vitamin C deficiency leads to Scurvy (unstable collagen molecules)

alpha-keto acid

Acids that have the keto group adjacent to the carboxylic acid

_______ define enzyme specificity

Active sites (lock and key model)

Coenzyme: ATP

Adenosine Triphosphate Functions as a substrate Protein covalent regulation Provides energy via transfer of phosphoric or nucleotide groups

Cofactors

Any nonprotein molecule or ion that is required for the proper functioning of an enzyme. Can be metal ions or vitamin-derived molecules (coenzymes) Cofactors can be permanently bound to the active site or may bind loosely with the substrate during catalysis.

Competitive reversible enzyme inhibition

Bind to active site Often have similar structure to substrate To reverse: Increase [S]

Uncompetitve reversible enzyme inhibition

Binds only to the ES complex but NOT at the active site CANNOT be reversed by substrate

Non-Competitive reversible enzyme inhibition

Binds to the E or ES complex but NOT at the active site The substrate can still bind, but the ESI complex will not yield products CANNOT be reversed by increasing [S]

How is substate specificity achieved in an enzyme?

By the interaction of the substate with the enzyme (Lock and Key model vs Induced Fit model)

Feedback Inhibition

Control of Protein Activity: Ligand Binding (negative feedback) Simple, 3-enzyme pathway End product of pathway acts as an inhibitor of the 1st enzyme (rate-limiting enzyme) Minimizes waste of intermediate products

Allosteric Regulation

Control of Protein Activity: Ligand Binding Regulatory molecules can modulate the activity of allosteric proteins by binding to distinct regulatory sites, separate from the functional sites Allosteric/Regulatory site: (different site; not the active site) pocket for binding of control molecules Allosteric inhibitors: modify the active site of the enzyme so that substrate binding is reduced/prevented Allosteric activators: modify the active site of the enzyme to increase its substrate affinity Allosteric regulators: ATP/CTP for ATCase Fru-2,6-P2 for PFK

Subcellular localization (compartmentalization)

Control of Protein Activity: Location Offers the possibility of selectively isolating metabolic processes and biochemical reactions that may interfere with each other Essential for cells to perform specialized biochemical functions, in particular those responsible for intracellular and intercellular signaling pathways

Extracellular trafficking

Control of Protein Activity: Location Proteins targeted to ER (ultimately to the cell membrane or the extracellular space) by signal sequences at the N-terminus of the protein

Drug Design: HIV

Drug Targets: Reverse transcriptase (RT) and Protease * RT inhibitors + Protease inhibitors = HIV Cocktail Reverse Transcriptase: HIV uses RT to convert its RNA into viral DNA (reverse transcription) Non-nucleoside reverse transcriptase inhibitors (NNRTIs) prevent HIV from replicating by blocking RT Protease: Protease inhibitors block protease (an HIV enzyme). By blocking protease, PI's prevent new (immature) HIV from becoming a mature virus that can infect other CD4 cells (peptidomimetic inhibitor)

Coenzymes: FMN and FAD

Flavin mono nucleotide & Flavin adenine dinucleotide Prosthetic groups Functions in redox reactions Vitamin source: B2 (riboflavin) Metabolic role: redox reactions involving 1 or 2 electron transfers (alternative participants in redox reactions; FADH2 is a weaker reducing agent than NADH)

Coenzyme: UDP-glucose

Functions as a substrate Metabolic role: transfer of glycosyl groups Most common glucose donor Glucose-1P + UTP = UDG-glucose + PPi (Formed by rxn driven by pyrophosphate (PPi) hydrolysis)

Coenzyme A (CoA)

Functions as a substrate Vitamin source: B5 (pantothenate) Metabolic role: transfer of acyl groups Carrier of acyl groups from glycolysis and beta-oxidation

Coenzyme: Tetrahydrofolate (THF)

Functions as substrate Vitamin source: B9 (Folate) Metabolic role: transfer of one-carbon substituents (especially formyl and hydroxymethyl groups). Provides the methyl group for thymine in DNA **For S-containing amino acids and nucleic acids

Dimerization of receptor proteins

Interaction of two receptor proteins to form a functional complex called a dimer

Enzyme inhibition: DIPF

Irreversible DIPF covalently binds to the hydroxyl group of a serine to inactivate the enzyme activity permanently

Enzyme inhibition: Iodoacetamide

Irreversible Iodoacetamide covalently binds to the sulfhydryl group to inhibit activity

Enzyme inhibition: Penicillin

Irreversible Penicillin is a potent inhibitor of glycopeptide transpeptidase

Lock and Key Model vs Induced Fit Model

LK = active site shape is rigid, only exactly complementary substrates can bind to form ES complexes IF = active site changes shape, the substrate binds to the active site - the active site changes shape so the substrate fits exactly forming an ES complex

Allosteric regulation via CTP of ATCase

Negative feedback loop via CTP At high concentrations of CTP, ATCase is allosterically inhibited, blocking the formation of product (N-carbomoylaspartate) The formation of this product is the 1st step in pyrimidine nitrogenous base synthesis which is eventually used to produce CTP, hence the negative feedback loop ATCase can be in the R state (active) or the T state (inactive). Allosteric regulation via CTP transforms ATCase into the T state

Coenzymes: NAD+ and NADP+

Nicotinamide adenine dinucleotide & Nicotinamide adenine dinuclotide phosphate) Functions in redox reactions Vitamin source: B3 (Niacin) Metabolic role: oxidation-reduction reactions involving 2 electron transfer NAD+ --> catabolic reactions NADP+ --> anabolic reactions

Allosteric Regulation via PALA of ATCase

PALA: potent transition state analog PALA is a ATCase inhibitor that blocks proliferation of mammalian cells in culture It mimics key features of ATCase to resemble the transition state that would normally catalyze the reaction

Drug Design: PFKFB3

PFKFB3 activity increases the rate of glycolysis in most cancer cells It has been found to be upregulated in numerous cells

Coenzyme: Vitamin K

Phyloquinone Prosthetic group Metabolic role: carboxylation of some glutamate residues **Blood clotting + quinone (electron carrier)

Iron-Sulfur Clusters (Fe-S)

Play a role in the electron transport chain Transport electrons through complexes I, II, and III to cytochrome C, before transfer to molecular oxygen

What methods are used to control protein activity?

Population Location Ligand Binding Covalent Modifications Environmental Factors

Control of Protein Activity: Population

Population controlled at the transcriptional and translational levels Turnover of protein via systematic proteolysis (proteolytic enzymes break down protein)

Coenzyme: Thiamine pyrophosphate (TPP/TDP)

Prosthetic group Vitamin source: B1 (Thiamine) Metabolic role: transfer of multi-carbon fragments containing a carbonyl group Used by carboxylase and decarboxylase Transfers (removes) CO2 from pyruvate to form acetaldehyde intermediate. NADH and H+ then come in and oxidize the acetaldehyde into ethanol (decarboxylation)

Coenzyme: Cobalamin

Prosthetic group Vitamin source: B12 (Cobalamin) Metabolic role: intramolecular rearrangements, transfer of methyl groups Structure related to heme but without one carbon in ring structure **Involved in enzymatic rearrangements: catabolism of odd-chain fatty acids, methylation of homocysteine, reductive dehalogenation

Coenzyme: Pyridoxal phosphate (PLP)

Prosthetic group Vitamin source: Vitamin B6 (pyridoxine) Metabolic role: Transfer of groups to and from amino acids (to form amino acids) Transaminase: PLP-dependent enzyme which catalyze the transfer of an amino group from an amino donor to an amino acceptor (allows transamination for AA synthesis) Lysine residue covalently binds PLP through Schiff base (mine) formation at the active site of transaminase

What mechanisms are used by enzymes to decrease the activation energy?

Proximity & Orientation Effects Transition State Preferred-Binding Acid-Base Catalysis Covalent Catalysis Cofactor-Assisted Catalysis

Thioredoxin (Trx) & Ferredoxin

ROS fighters Act as coenzymes in redox reactions Trx function in redox reactions: When you have an increase in ROS, you have an increase in disulfide bonds Trx holds intermediate with unwanted disulfide bond until it is fully reduced Trx reductase is the reducing agent Makes substrate with unwanted disulfide bond into (-SH)2 Ferredoxin function in redox reactions: Activated by light Acts as NADPH Used in oxidative phosphorylation Can hand over 1 electron at a time

ROS

Reactive Oxygen Species A group of extremely reactive peroxides and oxygen-containing radicals that may contribute to cellular damage

Catalysts _____ to enhance the reaction rate

Reduce the activation energy by stabilizing the transition state

Coenzyme: Vitamin A

Retinal Prosthetic group Metabolic role: vison/transcriptional control **Important signaling molecule

Coenzyme: SAM

S-adenosylmethionine Functions as a substrate Protein covalent regulation ATP derivative made from methionine and ATP Metabolic role: transfer of methyl groups (via highly reactive sulfonium group) Essential of protein methylation and DNA methylation

Control of Protein Activity: Location

Subcellular localization (compartmentalization) Extracellular trafficking Complexation with carrier proteins

Factors that affect the rate of an enzyme-catalyzed reaction

Temperature Decrease in ∆G (free energy change) [E] or [S] pH Presence of activators or inhibitors

The conversion of dUMP to dTMP

The conversion of deoxyuridine monophosphate to deoxythymidine monophosphate during DNS synthesis via methyl group transfer Facilitated by folate (THF) and Vitamin B12 (cobalamin)

Electron Transport Chain (ETC)

The portion of aerobic respiration that uses free oxygen as the final electron acceptor of the electrons removed from the intermediate compounds in glucose catabolism. Composed of four large, multiprotein complexes embedded in the inner mitochondrial membrane and two small diffusible electron carriers shuttling electrons between them. The electrons are passed through a series of redox reactions, with a small amount of free energy used at three points to transport hydrogen ions across a membrane. This process contributes to the gradient used in chemiosmosis. The electrons passing through the electron transport chain gradually lose energy. High-energy electrons donated to the chain by either NADH or FADH2 complete the chain, as low-energy electrons reduce oxygen molecules and form water. The end products of the electron transport chain are water and ATP. ** Electron Environmental Factors of Protein Control ** The ETC generates a proton gradient by pumping protons. This voltage drives generation of ATP. When voltage gets too high, the system gets paralyzed and instead of making ATP, it makes the ROS, superoxide.

Ligand-Binding

To communicate signal from outside to inside the cell Same signal can bind to diff receptors and affect diff responses in diff cell types Induces conformational change in receptor

Michaelis-Menten: kcat

Turnover number Number of substrate molecules converted into product per unit time at a single active site when the enzyme is fully saturated with substrate (# of conversions of substrate molecules converted into products at Vmax)

allosteric regulation of enzymes

may either inhibit or stimulate an enzyme's activity occurs when a regulatory molecule (activator or inhibitor) binds at a specific regulatory site on the enzyme and induces conformational or electrostatic changes that either enhance or reduce enzyme activity Allosteric enzymes do not conform to Michaelis-Menten kinetics Allosteric enzymes have multiple active sites which display cooperativity

Irreversible enzyme inhibition

permanently kill enzymes through covalent modifications

Michaelis-Menten: Vmax

reaction rate when the enzyme is fully saturated with substrate Vmax = kcat[E]T

What are the two classes of enzyme inhibitors?

reversible and irreversible

Prosthetic group

tightly-bound coenzyme Permanently bound thru covalent bond such as heme

Michaelis-Menten equation

v = (vmax [S])/(Km + [S])

What are two examples of Allosteric regulation?

via CTP of ATCase via PALA of ATCase

Signal Transduction

(Control of Protein Activity: Covalent Modifications (Phosphorylation)) A series of molecular changes that converts a signal on a target cell's surface to a specific response inside the cell Can be linear or cascade Cascade mechanism: Receptors activate downstream kinases, which then phosphorylate and activate their substrates until a response is achieved

Phosphorylation

(Control of Protein Activity: Covalent Modifications) Only occurs at the side chains of Serine, Threonine, and Tyrosine The amino acid nucleophilic group (-OH) attacks the terminal phosphate group on ATP, transferring the phosphate group to the amino acid side chain It is reversible **Ideal for signal transduction Examples: MAP Kinase pathway ICT Regulation GP Regulation

MAP Kinase Pathway (ERK Pathway)

(Control of Protein Activity: Covalent Modifications) (Phosphorylation & Signal Transduction) (Kinase activation in cell growth) A growth factor (or mitogen) binding includes dimerization of its receptor to cause mutual tyrosine phosphorylation, creating binding sites for adaptor molecules Grb2 (growth factor receptor-bound protein 2) and SOS (Son of sevenless, GEF) GEF replaces GDP in Ras (inactive state) with GTP, activating Ras. Ultimately, Ras is activated to cause activation of a kinase cascade of three mitogen-activated protein kinases (MAPKs): MAPKKK, MAPKK, and MAPK (Also called ERKs: extracellular signal-related kinases) The last kinase phosphorylates a number of different gene regulatory proteins

Glycogen phosphorylase (GP) regulation

(Control of Protein Activity: Covalent Modifications) (Phosphorylation & Signal Transduction) GP is a key enzyme in regulating the mobilization of glycogen in cells It cleaves glucose units from the non-reducing ends of glycogen Phosphorylation of T10 (Ser-14) by the enzyme PKA (via glucagon) converts GP forms (makes substate binding more accessible by moving the loop which sterically restricts access to the active site) Activation of GP degrades glycogen into glucose-1-phosphate and glycogen (glycogenolysis)

Isocitrate dehydrogenase (ICT) Regulation

(Control of Protein Activity: Covalent Modifications) (Phosphorylation & Signal Transduction) ICT is an enzyme in the tricarboxylic acid cycle (TCA) ICT is activated by ADP and NAD+ ICT is inhibited by ATP and NADH ICT's active site residues include Ser113, which is phosphorylated by protein kinase A (PKA) activated by cAMP via glucagon The phosphoryl group occupies almost the same location in the active site as the negatively charged portion of isocitrate The phosphorylated form of ICT is inactive because isocitrate binding is sterically blocked and electrostatically repelled the negative phosphoryl group Net Result: Decreased consumption of acetyl CoA from lipolysis in the TCA cycle for NAPH production Increased bypassing to oxaloacetate for gluconeogenesis

Secondary Signaling: GTP for Ras

(Control of Protein Activity: Ligand Binding) GTP binding proteins (G-proteins) function as molecular switches Ras is a small GTP binding protein GTP binding is facilitated by guanine-nucleotide exchange factors (GEFs) by orders of magnitude Ras is switched off by hydrolysis of the bound GTP This reaction is facilitated by the action of specific GTPase-activating proteins (GAPs) Anomaly in Ras function ---> cancer

Allosteric Regulation: ATP & CTP for ATCase

(Control of Protein Activity: Ligand Binding) Allosteric inhibition of ATCase by CTP Allosteric activation by ATP ATCase (aspartate transcarbamoylase) catalyzes the synthesis of N-carbamoylaspartate, the first intermediate in pyrimidine synthesis ATCase is feedback inhibited by CTP. ATP reverses this inhibition. CTP binds to R1 and R6 site of the R state ATCase; causing conformational change, converting ATCase to T state, making the active sites inaccessible to substrate ATP reverses this inhibition by binding to the same sites, balancing the concentrations of purines and pyrimidine nucleotide (ATCase back to R state; active state)

Secondary Signals

(Control of Protein Activity: Ligand Binding) Second messenger molecules relay signals received at receptors on the cell surface Examples: cAMP for PKA GTP for Ras

Ligand

A molecule that binds specifically to a receptor site of another molecule.

Sequential reactions

All substrates must bind to the enzyme before any product is released Types: Ordered & Random

Coenzyme: Vitamin E

Alpha-tocopherol ROS fighter ROS-reducing agent working together with Vitamin C (anti-oxidant coupled with vitamin C) Inactivates ROS before they can oxidize unsaturated membrane lipids and damage cell structures

Transition state

An unstable/maximum energy state during a reaction that must be achieved for the reaction to proceed It is not an intermediate - it its the moment where substates are turning into products

_______ + _______ = Holoenzyme

Apoenzyme (enzyme w/o cofactor) + Coenzyme = Holoenzyme (catalytically active)

Coenzyme: Vitamin D

Calciferol Transcriptional regulator for Ca++ (Ca++ control) Calcium absorption Increases protein activity

Reaction rate of enzyme-catalyzed reaction

Can be determined by monitoring the change in concentration of the reactants or the product as a function of time ∆[A] vs ∆[t]

Transamination

Combines lamination and deamination Mediates redistribution of amino groups among amino acids Uses PLP as a cofactor Reversible transfer of the amino group from an amine or an alpha-amino acid into an alpha-keto carboxylic acid

Types of reversible enzyme inhibition

Competitive Non-Competitive Uncompetitive

Reversible Inhibition effects on Km and Vmax

Competitive: No change Vmax Increase Km Non-Competitive: Decrease Vmax No change Km Uncompetitive: Decrease Vmax Decrease Km

Enzymes _______ the equilibrium constant of a reaction

DO NOT affect

Lineweaver-Burk Plot

Double reciprocal plot of Michaelis-Menten equation used to calculate Km and Vmax x int: -1/Km y int: 1/vmax

Enzymes affect the ________ of a reaction. HOW?

Enzymes affect the THERMODYNAMICS of a reaction by decreasing the activation barrier for both the forward and reverse reactions, causing them to speed up quickly Thermodynamics DO NOT tell us how fast or how the change occurs.

Control of Protein Activity: Ligand Binding

Inhibitors Allosteric regulators Secondary signaling

What is Ki?

Inhibitory constant The Ki of a drug can safely inhibit enzymes for functional use (high affinity)

Michaelis-Menten: Km

Michaelis Constant [S] at 1/2 Vmax

Electron carrier cofactors

NADH FADH2 Quinone Heme Fe-S

Types of bisubstrate reactions

Sequential reactions Double Displacement reactions

Quinone

Small, lipid-soluble, mobile electron carrier molecule

Ordered sequential reaction

Substrate(s) bind enzyme in defined sequence before any product is released

Heme

The prosthetic group of myoglobin and hemoglobin as well as other proteins Metabolic role: electron transfer

Steady State Kinetics

The study of enzymatic reaction rates in which the rate of ES formation and breakdown are equal.

Lipid soluble vitamins

Vitamins A, D, E, K

The reaction will continue if: (Steady state kinetics)

[S] >> [E]

Dimer

a molecule or molecular complex consisting of two identical molecules linked together.

What is an enzyme

a protein (or protein-based molecule) that speeds up a chemical reaction in a living organism It acts as a catalyst for specific chemical reactions, converting a specific set of reactants (substates) into specific products

Random sequential reaction

addition/release of substrates and products is random

Guanine-nucleotide exchange factor (GEF)

an SOS protein that triggers the release of GDP from the Ras protein, thereby permitting Ras to acquire a molecule of GTP Results in an increase in RAS in the active GTP-bound form

Kinase

an enzyme that catalyzes the transfer of a phosphate group from ATP to a specified molecule.

Enzymes have:

high specificity for their substrate and the reactions they catalyze high reaction rates

Allosteric Regulation: Fru-2,6-P2 for PFK

(Control of Protein Activity: Ligand Binding) Allosteric activation of PFK (glycolysis activation) by Fru-2,6-P2 PFK (phosphofructokinase) is a very important regulatory enzyme of glycolysis (allosteric enzyme) It catalyzes the "committed" step of glycolysis (conversion of fructose-6-phosphate (F6P) to fructose 1,6-biphosphate) PFK is allosterically inhibited by ATP The most potent activator is Fru-2,6-P2 (fructose 2,6-biphosphate) Abundance of F6P causes higher concentration of Fru-2,6-P2. The binding of Fru-2,6-P2 increases affinity of PFK for FP6 and diminishes the inhibitory effect of ATP.

Secondary Signaling: cAMP for PKA

(Control of Protein Activity: Ligand Binding) cAMP (the secondary messenger) plays roles in cellular responses to many hormones and neurotransmitters Protein kinase A (PKA) is a main effector of cAMP It is activated by the binding of cAMP to two sites on each of PKA's two regulatory subunits This releases the active catalytic subunits of PKA

Double displacement bisubstrate reaction

(Ping pong reaction) One or more products are released before all substrates bind the enzyme Enzyme is temporarily modified (intermediate) The substrates appear to bounce on/off the enzyme like a ping pong ball

Control of Protein Activity: Covalent Modifications

(Post-translational modifications) Phosphorylation Ubiquitinylation Proteolysis