Biochem Exam 2 list of topics

Enzymes

Catalysts

• enzymes are biological catalysts

• increase the rate of a reaction

• mostly proteins, some are catalytically active RNA

• most enzyme names end in -ase

Activation Energy/Diagrams

• activation energy is how much energy must be put into a reaction for it to occur

• enzymes work by decreasing the activation energy

  • do this by stabilizing the transition state of the molecule - the highest energy species in the reaction pathway

Free energy (Gibbs free energy change)

• Gib’s free energy (ΔG) shows whether a reaction with be spontaneous or not

• enzymes have no effect on ΔG

• ΔG < 0 is an exergonic reaction - will be spontaneous, ΔG > 0 is endergonic reaction, which needs energy input to occur

• tells about whether or not reaction is spontaneous, NOT the speed of the reaction

• a reaction at equilibrium has a ΔG=0

• standard free energy change (ΔGº’) of a reaction is related to the equilibrium constant (K’_eq)

  • ΔGº’ = -RTlnK’_eq

  • as K’_eq goes up, ΔGº’ goes down

• reaction equilibrium is determined by the free energy difference between products and reactants, enzymes cannot alter this difference

Active Site

• small - contributes a small portion of the enzymatic volume

• 3D crevice created by amino acids from the primary structure

• place where enzyme non-cavalently bonds to substrate

• create unique microenvironments

• enzyme specificity depends on the molecular architecture of the active site

• may include distinct residues

  • residues may be far apart on the linear view of the structure, but close to each other in the 3D structure

Binding Models:

1. Lock and Key - if the enzyme is the right shape for the substrate to fit into, they will click together

2. Induced Fit - more accurate/higher level explanation - the substrate may not fit perfectly into the “raw shape” of the enzyme, but once they bond the enzyme will change shape so that they fit perfectly   

• interaction of the enzyme and the substrate at the active site involves multiple weak interactions

Cofactors

• small molecules that some enzymes require for activity

• can be coenzymes (organic molecules derived from vitamins) and metals

• tightly bound coenzymes are called prosthetic groups

• enzyme w/ cofactor = holoenzyme

• enzyme w/o cofactor = apoenzyme   

Enzyme Kinetics

• study of reaction rates - measure velocity as a function of substrate concentration with a fixed amount of enzyme

• reaction velocity vs. substrate concentration (how they are graphed)

• $E+S\leftrightarrow ES\leftrightarrow E+P$

  • E+S→ES is k1

  • ES→E+S is k-1

  • ES→E+P is k2

  • E+P→ES is k-2

  • k1 and k-1 are binding

  • k2 and k-2 are catalysis

Rate and Velocity

• at steady state - rate formation of ES = rate of breakdown of ES (k1=k-1)

Michaelis-Menten Plot/Equation

• V₀=V_max ( [S] ÷ ([S]+K_m) )

Km

• michaelis constant - K_m=(k-1 + k2)÷k1

  • when more enzymes are binding to substrate rather than releasing substrate (k1>k-1) K_m is small

• when V₀=1/2V_max, K_m=[S]. thus, K_m is the substrate concentration that yields ½ V_max

• reflection of the affinity of enzyme for its substrate /.measure of ES binding affinity

• constant for any given substrate/enzyme pair

• small K_m means tight ES binding, large K_m means weak binding

Vmax

• the maximum velocity a reaction can happen

• plateu of a michaelis-menton plot

• happens becuase if all the available enzyme is being used, more substrate will not help speed up the reaction

Kcat, catalytic constant, turnover number

• how fast the ES complex proceeds to E+P

• number of catalytic cycles that each enzyme active site undergoes per a unit of time

• rate constant of the reaction WHEN enzyme is saturated with substrate

• first order rate constant (sec-1)

• turnover number is the product formation per unit enzyme at saturated [S] ← so same thing as K_cat

• also known as k2 from the enzyme kinetics equation

kcat/Km, catalytic efficiency

• reflects both binding and catalytic events - indicates how velocity of reaction varries according to how often the enzyme and substrate combine

• best value to represent the enzymes overall ability to convert substrate to product

• efficiency increases as K_cat increases or K_m decreases

• useful for:

  • substrate specificity

  • comparison of catalytic efficiencies

Lineweaver-Burk Plot/Equation

• 1/V₀ = K_m/V_max • 1/S + 1/V_max

  • slope of the line is K_m/V_max

  • x-intercept is -1/K_m

  • y-intercept is 1/V_max

Inhibition of Enzymes: competitive, non-competitive, uncompetitive

• irreversible

  • covalent bonds

  • cyanide, nerve gas, pesticides

    • nerve gas inhibits AChE

  • often slow

• reversible

  • characteristics:

    • non-covalent bonds

    • often VERY rapid

    • kinetically distinct

    • medical applications

  • 3 types

    • competitive

      • binds to the same place on the enzyme the substrate would bind - block the substrate from forming ES complex

      • increases K_m (competition reduces substrates affinity for enzyme)

      • does not change V_max - inhibitor can be outcompeted by the substrate, inhibitor does not actually “harm” enzyme

      • one example is statins - control cholesterol biosynthesis, ex: atorvastatin (lipitor) and simvastatin (Zocor)

    • non-competitive

      • can bind to enzyme or ES complex, substrate can also bind to EI complex

      • binds to a site the subtrate does NOT bind to

      • K_m does not change (since substrate can bind to inhibiteed enzyme, binding affinity doesn’t change)

      • V_max decreases

    • uncompetitive

      • binds to the ES complex preventing it from becoming E+P

      • decreases K_m (locking of ES together simulates tight binding which is low K_m)

      • decreases V_max because some enzymes will be permanantly stuck as ESI

Regulation of Enzyme Activity

Allosteric control

• a molecule that is different than the substrate binds to the enzyme and tells it to function (or not to function)

• example: aspartate transcarbamoylase (ATCase) catalyzes the first step in pyrimidine synthesis, and the end product of the pathway is CTP. CTP can bind to ATCase at a regulatory or allosteric site and inhibit the enzyme, preventing the production of more CTP

  • ATCase has an R state and a T state

    • PALA binds and causes structural changes that convert the T state into the expanded, active R state - PALA functions as a homotropic effector

      • PALA also allows scientists to study the R state of ATCase

    • T state has low affinity for substrate/low catalytic activity - R state is active form

    • T state is favored in absence of substrate, two states are in equilibrium

    • binding of substrate disrupts equilibrium in favor of R state - COOPERATIVITY

  • effect of inhibitors on allosteric enzymes is called heterotropic effect

  • allosteric interactions in ATCase are mediated by large changes in quaternary structure

• creates a negative feedback mechanism

• these enzymes will NOT follow Michaelis-Menten kinetics

  • instead they display sigmoidal curves because of cooperation between subunits

Multiple forms of enzymes

• isoenzymes or isozymes are enzymes that are encoded by different genes

  • catalyze the same reaction, but may display different regulatory properties

  • may be expressed in a tissue-specific or developmentally specific patter

  • appearance of certain isozymes in the blood is a sign of tissue damage

    • example: lactate dehydrogenase has four subunits that make up five types (LDH-5 through LDH-1) the composition of the subunits can change depending on age

Reversible Covalent Modifications

• enzymes can be modified by the covalent attachment of a molecule

• phosphorylation and acetylation are common modifications

• most covalent modifications are reversible

• protein kinases modify proteins by attaching a phosphate to a serine, threonine, or tyrosine residue

  • ATP serves as the protein donor

• protein phosphatases remove phosphates added by kinases

• example: cyclic AMP activates protein kinase A by altering the quaternary structure

  • cAMP stimulates PKA by binding to PKA’s regulatory (R) subunits causing them to dissociate from the catalytic (C) subunits, the free C subunits are the active form

  • a “pseudosubstrate” sequence of the R subunit blocks the active site of the C subunite when the subunits ate bound to eachother

  • mutations in PKA can cause Cushing’s syndrome

Proteolytic activation, Zymogens

• enzymes can be made in a pre-enzyme form called a zymogen or proenzyme

• the zymogen is activated by proteolytic cleavage into the active form

• example: chymotripsinogen

  • chymotripsinogen is the inactive form, cleaved between 15 and 16 by trypsin to make pi-chymotrypsin (active) then cleaved again into an A, B, and C chain (1-13, 16-146, and 149-245) which together are alpha-chymotrypsin (also active)

• can be thought of as one of the plastic pull tabs in a battery compartment, has everything it needs to work but you have to remove the tab to activate it

Induction/Repression: Controlling the amount of enzyme present

??? no slides on this?

Lipids

• lipid = biomolecule that is soluble in nonpolar solvents

Function of lipids

• structural molecules

• dietary consituents and metabolic fuels

• insulation

• signaling

• digestive aids

• pulmonary surfactant

• buoyancy (for marine mammals)

• production of water (metabolic water)

• antioxidants

Classes of Lipids

• simple lipids - esters of fatty acids with various alcohols

  • fatty acids

    • CH₃(CH₂)_nCOOH

    • carboxylic acid (COOH) at one end and methyl group (CH₃) at the other

    • numbered beginning with the carboxyl terminal carbon atom, then 2, 3, - or alpha carbon is the first carbon not in the carboxyl group

    • methyl group carbon is the omega (ω) carbon

    • 4 or more carbons, but always an even number

    • pKa is about 4.5

    • short chain length or double bonds will make a fatty acid more fluid

  • glycerides - ester(s) of fatty acid(s) with glycerol

    • esters formed from glycerol backbone and fatty acids that are very hydrophobic

      • monoacylglycerol has one fatty acid

      • diacylglycerol has two

      • triacylglycerol has three

  • waxes - esters of fatty acids with long chain of alcohols

• compound lipids - esters of fatty acids with alcohol and another group - maybe also known as polar (complex) lipids? 

  • phospholipids - lipids containing phosphate in addition to fatty acid and alcohol

  • glycolipids (glycosphingolipids) - containing a fatty acid, sphingosine, and carbohydrate

• derived lipids - produced in hydrolysis of the first two groups, or they are present in association with them in nature

  • examples

    • steroids

    • carotenoids

    • alpha-tocopherol

  • hydrocarbons - contain only C and H

    • beta-carotene

  • long chain alcohols

    • sterols (cholesterol, phytosterols, tocopherol)

      • cholesterol is a lipid based on steroid nucleus

      • modified on one end by the attachment of a fatty acid chain and at the other end by a hydroxyl group

      • im membranes, hydroxyl group interacts with phospholipid head groups

Saturated vs. Unsaturated Fatty Acids

• saturated - only single bonds, “saturated” with hydrogens on all the carbons, can be more tightly packed and lead to a less viscous membrane

• unsaturated - has some double bonds, presence makes the membrane more fluid

Viscosity and Fluidity of Fatty Acid Chains

• short chain length or double bonds will make a fatty acid more fluid

Triacylglycerols (TAGs)

• a type of glyceride, glycerol backbone and three hydrophobic fatty acid tails

Naming Fatty Acids/Triacylglycerols

• ??????

Phospholipids

• phospholipids - lipids containing phosphate in addition to fatty acid and alcohol

  • four components: one or two fatty acid tails, a platform (glycerol or sphingosine), a phosphate, and an alcohol

  • glycerophospholipids (phosphoglycerides) - most abundant lipids in cell membranes, composed of glycerol, two fatty acids, phosphate, and an alcohol

    • main component of biological membranes

    • main four found in membranes are:

      • phosphatidylserine (has COO- and NH3 groups)

      • phosphatidylcholine (has N+ with three methyl groups on end)

      • phosphatidylethanolamine (has an NH3 on the end)

      • phosphatidylinositol (has a lot of OH and distinct net-like structure)

  • sphingomyelin - sphingolipids with phosphate

    • found in cell membranes, especially in membranous myelin sheath

    • a phospholipid but also a sphingolipid

    • sphingosine backbone - contains long, unsaturated hydrocarbon

    • sphingosine+fatty acid (amide bond) = ceramide

    • ceramide + phosphocholine = sphingomyelin

Glycolipids

• glycolipids (glycosphingolipids) - containing a fatty acid, sphingosine, and carbohydrate

  • sphingosine backbone (a sphingolipid)

  • sphingosine + fatty acid (amide bond) = ceramide

  • polar head group = sugar

    • cerebrosides with glucose as polar head group are the most simple glycolipids

  • blood group antigens on cell surface of red blood cells are glycolipids

    • O antigen has no extra chain, A has an extra GalNAc, B has an extra Gal

Sphingolipids

• any lipid with a sphingosine backbone (i think)

Biological Membranes

Fluid Mosaic Model

• mosaic of globular proteins and phospholipid bilayer form the cell membrane

• the membrane is viscous, but fluid - “liquid crystal”

• lipids and proteins are free to move laterally

Membrane Proteins

• 30% peripheral (extrinsic)

• 70% integral (intrinsic)

• also can be amphitropic

• ratio of protein to lipid in different membranes vary:

  • myelin (1:4)

  • plasma membrane (1:1)

  • inner-mitochondrial membrane (4:1)

  • thylakoid membrane (4:1) - in chloroplast of plant cells

Integral

• buried in the membrane

• one or more membrane-spanning helix (20 amino acids or 30 Å)

• difficult to dissociate from the lipid

• insoluble in aqueous solution

• specific lipid requirements for function

• example: NKA

Peripheral

• exposed at one surfae

• electrostatic, H bond, hydrophobic (lipid) anchor

• may be bound to an integral protein

• easy to dissociate from the membrane

• soluble in aqueous solution

• example: cytochrome c (binds to cardiolipin)

Amphitropic

• can be bound to the membrane peripherally or free floating

• example: CCT

  • CCT is CTP:PC cytidyltransferase, and it controls phosphatidylcholine synthesis

  • interconversion between soluble (inactive) and insoluble (active)

  • membrane binding domain (M domain) is a long amphipathic helix that responds to physical changes in phosphatidylcholine deficient membranes

  • dephosphorylation of the membrane-bound form stabilizes membrane binding

Membrane Fluidity

• fluidity is the opposite of viscosity

• correlated with the magnitude and frequency of movement

• higher temp = higher fluidity

Trans/Gauch isomerization

• molecular characterization of the gel phase vs. liquid phase of a lipid

  • liquid phase is gauche isomerization - not in a lineart arrangement, harder to pack together, makes the membrane more fluid - higher potential energy

  • gel phase is trans isomerization - linear arrangement allows for tighter packign and stiffer membrane - lower potential energy

Homeoviscous adaptation

• “goldilocks” idea

  • if a membrane is too stiff it will be too easily broken

  • if a membrane is too fluid it will be too crumbly - will just fall apart

Effect of Chain Length

• shorter chain length allows fatty acids to move more easie

Effects of Saturation/Unsaturation or cholesterol

• unsaturated fatty acids result in a more liquid membrane - more likely to be seen in lower temperatures

• saturated fatty acids cause a more viscous membrane - more likely to be seen in higher temperatures

• cholesterol stiffens the membrane

  • cholesterol is essential and most abundant neutral lipid in the membrane

  • 90% of cellular cholesterol is associated with the plasma membrane

  • amphipathic

  • restricts motion of acyl chains

  • hydrogen bonding from cholesterol stabilized fluid phase membranes, condeses packing, and increases membrane thickness

  • selective association with polar lipids - longer chain, saturated tails (sphingolipids and phosphotidulcholine)

  • modulates activity of membrane proteins like the sodium potassium pump

  • can also form lipid microdomains

Lipid Bilayers

Movement of Lipids & Proteins in Membranes

• roatation - just twirling around

• lateral - switching with its neighbors

• transverse - switching leaflets of the bilayer

Membrane asymmetry

• transverse asymmetry says that some lipids will be more prevelent in the outer monolayer (ie phosphotidyl choline and sphingomyelin) and some are more prevelent inside (phosphatidyl ethanolamine and phosphatidyl serine)

• some lipids, if seen on the “wrong side” of the membrane are a signal for apoptosis to occur

Flippase, Floppase, Scramblase

• flippase

  • catalyze the movement of specific phospholipid species from the extracellular leaflet to the cytosolic leaflet

  • use ATP

  • examples:

    • type-IV P-type ATPases, P4-ATPases

• floppase

  • catalyze the movement of specific phospholipid species from the cytosolic leaflet to the extracellular leaflet

  • use ATP

  • examples:

    • ABC-transporters

• scramblase

  • bidirectional, no energy required, not specifc

  • Ca2+ dependant

Lipid rafts

• a form of lateral asymmetry

• plasma membrane microdomains, enriched in cholesterol and sphingolipids

• lateral comparmentalization

Caveolae

• a form of lateral asymmetry

• subdomain of lipid rafts

• caveolin-1 enriched

• functions of microdomains: segregate or concentrate membrane proteins to organize signal transduction paths, immune responses, folic acid uptake, transcytosis

Membrane Function

Cellular Metabolism

• ATP is the energy currency of life

• ATP can be formed by the oxidation of carbon fuels

• metabolic pathways are highly regulated

Catabolism

• degradative pathway - generation of energy from macronutrients

• formation of NADH, FADH2, and ATP

  • NADH and FADH2 are used to make ATP

• ATP-generating reactions

Anabolism

• biosynthesis

• synthesizing molecules that make up the cell

• use NADH, FADH2, or ATP

• ATP utilizing reactions

Metabolic Pathways - Three Stages, Compartmentalization

• compartmentilization allows:

  • separate pools of metabolites in a cell

  • simultaneous operation of opposing metabolic paths

  • high local concentrations of metabolites

  • coordinated regulation of enzymes

    • example: fatty acid synthesis enzymes are in the cytosol, breakdown enzymes are in the mitochondria

Substrate Level Phosphorylation

• direct transfer of a phosphate group - like in reactions that use ATP for enegy

Oxidative Phosphorylation

• use of energy from FADH2 and NADH to make ATP - like in electron transport chain

• creates more ATP than substrate level, but also requires oxygen

Thermodynamics -Free Energy

• reactions can happen spontaneously is the change in free energy (ΔG) is negative

Coupling Reactions

• if a reaction is not favorable energetically, it can be coupled with a favorable reaction to make it more favorable

• example:

  • A <> B + C      ΔG=+21 kJ/mol

  • B <> D            ΔG=-34 kJ/mol

  • A <> C + D      ΔG=-13 kJ/mol

    • ends up being spontaneous because the reactions are coupled

ATP - phosphoanhydride Bonds

• ATP is main source of energy for the body

• not stored - rapidly used and remade

• common to organisms that do and do not use oxygen

• phosphate group held on with phosphoanhydride bonds

  • energy rich - release energy when broken

  • Gibs free energy for ATP hydrolysis is about -50kJ/mol

• ortho-phosphate cleavage is removal of 1 phosphate

  • ATP → ADP + Pi

• pyro-phosphate cleavage is removal of 2 phosphates

  • ATP → AMP + 2Pi

Glycolysis –

Degradative, Cytoplasm

Fates of Pyruvate

Overall Reaction – 10 steps

Stage 1 – energy investment

• 1 moles of ATP are consumed for each mole of glucose

• glucose is converted to fructose 1,6-bisphosphate

• glucose is trapped inside teh cell and at the same time converted to an unstable form that can be readily cleaved into 3-carbon units

• 6C + 2ATP → 6C-2Pi

Stage 2 – lysis and energy production

• 2a. Lysis

  • F-1,6-BP is cleaved into two 3-carbon units of glyceraldehyde-3-phosphate

  • 6C-2Pi → 3C-Pi + 3C-Pi

• 2b. Energy Generation

  • glyceraldehyde-3-phosphate is oxidized to pyruvate

  • 4 moles of ATP and 2 moles of NADH are gained from each initial mole of glucose

  • (3C-Pi)2 + 2Pi + 4ADP → (3C)2 + 4ATP

Structure (Glucose, Pyruvate)

• glucose - C6H12O6

Enzymes - Types and Names

• hexokinase

• phosphoglucose isomerase

• phosphofructokinase

• aldolase

• triose phosphate isomerase

• glyceraldehyde 3-phosphate dehydrogenase

• phosphoglycerate kinase

• phosphoglycerate mutase

• enolase

• pyruvate kinase

Ethanol Metabolism / Lactic acid fermentation

• ethanol metabolism - pyruvate —pyruvate decarboxylase→ acetaldehyde —alcohol dehydrogenase→ ethanol

• lactic acid fermentation - pyruvate —lactate dehydrogenase→lactate

Energy charge

• energy charge regulates metabolism

• is an index used to measure the energy status of biological cells

Glycolysis regulation: Muscle

Phosphofructokinase: ATP, AMP, low pH

• phosphofructokinase is the most important regulator in mammals

• does the first “commited” step in glycolysis - F6P + ATP → F1,6-BP + ADP

• ATP binding lowers PFK affinity for F6P

• AMP reverses inhibitory action of ATP

• PFK activity increases when there is a lower ATP/AMP ratio (when energy charge falls)

• low pH inhibits PFK activity by enhancing inhibitory activity of ATP - such as drop of pH because of lactic acid during exercise

  • inhibition protects the muscles from damage that would result from the accumulation of too much acid

• allosteric regulation - each of the 4 subunits of PFK have an active site for F6P and an allosteric site for ATP

Hexokinase: glucose 6-phosphate, negative feedback

• allosterically inhibited by its product glucose 6-phosphate

• when there is enough product hexokinase is inhibited - negative feedback

Pyruvate kinase: ATP, AMP, F 1,6-BP, feedforward stimulation

• allosterically inhibited by ATP and stimulated by F 1,6-BP (by PFK)

• F 1,6 BP is made early in the glycolysis pathway, activates pyruvate kinases before it is needed - feed forward stimulation

Glycolysis regulation: Liver

Phosphofructokinase: ATP, citrate

• PFK is a sensor of the energy level

• inhibited by citrate

• higher energy charge results in a lower citric acid cycle flux, since glycolytic flux is matched higher energy charge also results in lower glycolytic flux

PFK2/FBPase2, bifunctional enzyme: F2,6-BP → PFK

• PFK2 catalyzes F-6P→F-2,6-BP, which activates PFK to turn F-6P→F-1,6-BP and promotes glycolysis

• FBPase2 turns F-2,6-BP back into F-6P, and since there is no PFK stimulation - inhibits glycolysis

PFK2: insulin

• PFK2 helps turn F6P→F2,6BP which stimulates PFK stimulating glycolysis

PKA: glucagon

• PKA phosphorylates PFK2 deactivating it, allowing FBPase2 to be active, which turns F2,6-BP into F6P and limits glycolysis

Glucokinase: 50x less sensitive, low affinity for glucose, not inhibited by G6P

• glucokinase is the hexokinase isoenzyme found in the liver

• role is to provide glucose 6-phosphate for the synthesis of glycogen and for the formation of fatty acids

• lower affinity for glucose than other hexokinases, operates only when glucose is abundant

• hexokinase is inhibited by G6P, but glucokinase is not - allows it to continuously process and store glucose when levels are high enough, unlike other cells that stop themselves from “hoarding” energy

Pyruvate kinase: ATP, AMP, F 1,6-BP, feedforward stimulation

• regulated allosterically - like it is in muscle

• also regulated by covalent modification

• low blood glucose leads to the phosphorylation and inhibition of liver pyruvate kinase

  • glucagon triggers this phosphorylation, prevents the liver from consuming glucose when it is more needed in other parts of the body

Glycolytic Flux

Exam Format

• On everything from the end of last exam (enzymes) to Lecture 19 Slide 12

• similar to Ex 1

• 9 T/F (1 pt each)

• 10 fill in blank (2 pt each)

• 4 decrease, increase, no change

• 17 multiple choice

• 2 short essay - probably sometimes names or write answers less than 2-3 sentences