Heme Synthesis, Enzymes and Metabolism

• The initial pages are an overview of heme synthesis and degradation, enzymes, and metabolism
• A diagram shows a mitochondrion with labels for key structural components
• The mitochondrion supports fatty acid oxidation, the Krebs cycle (CAC), and electron transport with ATP synthase
• The inner membrane is the site of electron transport and ATP synthesis
• The outer membrane is permeable, and its permeability is facilitated by porins

Heme Composition and Function

• Heme is made in the liver and erythroblasts (red blood cell precursors)
• It consists of protoporphyrin IX, iron (Fe), and four nitrogen atoms
• Porphyrins are constructed from four molecules of a monopyrrole derivative
• Porphobilinogen is a precursor in porphyrin synthesis
• Iron is inserted into the protoporphyrin ring by an enzyme
• Iron has a high binding affinity for protoporphyrin
• Heme acts as a prosthetic group for proteins involved in oxygen transport and storage, O2 metabolism, antioxidation, and electron transport
• Heme emits red fluorescence under UV light

Heme Biosynthetic Pathway

• Consists of seven steps that occur in the mitochondria and cytoplasm
• Glycine serves as a major precursor

Step 1: Making δ-aminolevulinate (ALA)

• ALAS (aminolevulinate synthase) catalyzes the condensation of succinyl-CoA and glycine
• This is the rate-limiting and highly regulated step of heme biosynthesis
• Requires the cofactor pyridoxal phosphate (PLP)
• ALAS is transported to the mitochondria
• Heme inhibits ALAS synthesis and transport

Step 2: Formation of Porphobilinogen

• Porphobilinogen synthase catalyzes the reaction
• Water is released as a by-product

Step 3: Assembly of Porphyrins from Porphobilinogen

• Catalyzed by porphobilinogen deaminase
• Four porphobilinogen molecules are linearized
• Forms methylene bridges between monopyrroles
• Releases ammonium ions as a by-product

Step 4: Formation of Uroporphyrinogen

• Uroporphyrinogen III synthase catalyzes the reaction
• Cyclizes the linear tetrapyrrole to make an asymmetric molecule
• Releases water as a by-product

Steps 5-7: Further Modifications of the Porphyrin Structure

• Methyl groups are added to the molecule
• Vinyl groups are added
• Ferrous iron (Fe2+) is added by ferrochelatase

Porphyrias

• Genetic disruptions in the heme biosynthetic pathway, occurring in erythroblasts or the liver
• Acute intermittent porphyria affects the liver, leading to severe abdominal pain and neurological dysfunction
• Congenital erythropoietic porphyria causes teeth to fluoresce red, red-stained urine, sensitivity to sunlight, and can cause anemia

Heme Degradation

• Hemoglobin must be degraded when red blood cells lyse
• Degradation protects cells from oxidative damage
• Iron is made available for storage or other uses
• Humans possess at least three isozymes of heme oxygenase (HO)
• High heme concentrations in the body can be damaging
• Proteins are proteolyzed, but not the heme
• Results in the production of carbon monoxide (CO)
• Bilirubin is an important antioxidant that is highly abundant and hydrophobic
• Jaundice can occur if bilirubin levels outstrip serum albumin solubility, so it must bind to serum albumin to be transported from the liver
• Newborns are checked for bilirubin levels, and if levels are too high they are treated with UV light

Enzymes

• Enzymes are proteins, but sometimes also RNA, that act as catalysts
• They stabilize the transition state, which is the highest energy species in the reaction pathway
• Enzymes act on reactants, which are also known as substrates
• Enzymes are highly specific due to a precise interaction between the enzyme and the substrate
• Trypsin and thrombin have different degrees of specificity for their substrates

Enzyme Classes

• Oxidoreductases: Transfer electrons (hydride ions or H atoms) through oxidation-reduction reactions
• Transferases: Catalyze group transfer
• Hydrolases: Catalyze hydrolysis (transfer of functional groups to water)
• Lyases: Catalyze cleavage of C—C, C—O, C-N, or other bonds by elimination, leaving double bonds or rings, or addition of groups to double bonds
• Isomerases: Catalyze the transfer of groups within molecules to yield isomeric forms
• Ligases: Catalyze the formation of C-C, C-S, C-O, and C-N bonds by condensation reactions coupled to the cleavage of ATP or a similar cofactor
• Translocases: Catalyze the movement of molecules or ions across membranes or their separation within membranes
• Proteases catalyze by breaking bonds
• Catalyze bind covalently or noncovalently to the enzyme, but with a negligible dissociation constant.

Cofactors

• Some enzymes need cofactors to operate
• Cofactors are small molecules that some require activity
• Coenzymes and metals are the two primary categories of cofactors
• Coenzymes that bind tightly are called prosthetic groups
• A holoenzyme is an enzyme with its cofactor
• An apoenzyme is an enzyme that lacks its cofactor

Enzyme Regulation

• Allostery: Reversible, noncovalent binding of allosteric modulators or allosteric effectors
• Allostery causes changes in the 3D structure of the enzyme and therefore affects catalysis
• Reversible covalent modification
• Binding of separate regulatory proteins
• Proteolytic cleavage
• Covalent modification (adding or removing chemical groups)
• When chemical resources are plentiful, cells can synthesize and store glucose and other metabolites
• When chemical resources are scarce, cells use these stored resources to fuel cellular metabolism
• The availability of specific catalysts allows for the regulation of these reactions
• Allosteric enzymes are regulated by homotropic or heterotropic binding

Regulatory Binding Types

• Homotropic - Regulation in which the substrate and modulator are identical
• Heterotropic - Regulation in which the modulator is a molecule other than the substrate
• ATP is a positive regulator, while CTP is a negative regulator

Aspartate Transcarbamoylase

• Catalyzes the formation of carbamoyl aspartate, an early step in pyrimidine biosynthesis
• ATP acts as a positive regulator; CTP acts as a negative regulator

Phosphorylation

• Protein kinases catalyze the attachment of phosphoryl groups to specific amino acid residues (Ser, Thr, Tyr, His)
• Phosphoprotein phosphatases remove phosphoryl groups from the same target proteins
• Multiple phosphorylation sites within a protein can offer subtle modulation of enzyme activity

Proteolytic Cleavage

• Proteins are broken down into smaller peptides or amino acids by proteases
• Zymogens are inactive precursors that are cleaved to form an active protease enzyme

Gibbs Free Energy & Enzymes

• If ∆G is negative, the reaction is exergonic and spontaneous
• At equilibrium, ∆G = 0
• Endergonic reactions require energy input and will not occur spontaneously
• The ∆G of a reaction provides no information about the rate of the reaction
• The ∆G of a reaction only depends on the free energy difference between reactants and products
• ∆G is independent of how the reaction occurs
• A reaction can differ from its ∆Go depending on the concentrations of the reactants and products
• Reactions proceed through a transition state and form something that is no longer a substrate but also not yet a product
• Enzymes lower the activation energy of a reaction and speed it up

Enzyme-Catalyzed Reactions

• Rate constants not affected by reaction conditions do not equal reaction rate (affected)
• Enzymes bring substrates together to form an enzyme-substrate complex, which occurs on the active site on the enzyme
• E+S<->ES->E+P

Induced Fit Model of Enzyme-Substrate Binding

• Binding energy is the free energy released upon interaction of the enzyme and substrate
• Binding energy is greatest when the transition state is formed, creating larger numbers of interactions than when the enzyme is bound to the substrate
• In an ES complex, the substrate is not fully complementary to the binding site

Features of Enzyme Active Sites

• The active site is a three-dimensional cleft or crevice created by amino acids from different parts of the primary structure
• The active site constitutes a small portion of the enzyme volume
• Active sites create unique microenvironments
• The interaction of the enzyme and substrate at the active site involves multiple weak interactions
• Enzyme specificity depends on the shape and chemistry of the active site at the active site

Multiple Substrate Reactions

• Sequential reactions are characterized by the formation of a ternary complex consisting of both the enzyme and both substrates (ordered or random)
• Double-displacement reactions are characterized by the formation of a substituted enzyme intermediate

Enzyme Kinetics & Formulas

• Initial velocity (Vo): when P=0, units of conc. per time
• MM equation: Vo=Vmax[S]/[S]+Km
• Steady-state assumption: Rate of formation of ES = Rate of breakdown of ES
• Km is the [S] at ½ Vmax and describes substrate affinity
• High Km means low substrate affinity; low Km means high substrate affinity

Enzyme Kinetics Experiment

• As substrate conc increases, rxn velocity increases
• Lineweaver-Burk plot is used
• Km = -1/x-intercept
• Vmax = 1/y-intercept
• Kcat Turnover number of the number of the enzymes, turnover represents number of substrate molecules converted into product per time in s
• Vmax=kcat(Et)
• Kcat/Km measure of catalytic efficiency, rxn rate directly proportional
• Michealis Menten Kinetics is not obeyed by allosteric enzymes

Carbonic Anhydrases

• Enhance reaction rates
• Plays a role in the generation of aqueous humor of the eye
• Lack of activity causes osteopetrosis and intellectual disability
• Human Carbonic Anhydrase II and its Zinc Site
• The Zn2+ is bound to four ligands: Bound to histidine residues and bound to either water molecule or a hydroxide ion
• Histidine proton shuffle, deprotonates easily using a general base in cytosol

Inhibition of Enzymes and Km

• Irreversible enzyme inhibitors which bind covalently or noncovalently to the enzyme, but with a negligible dissociation constant
• Reversible: rapid dissociation to inhibitor complex
• Km is the substrate concentration in the blood -Low km: means a high affinity of the enzyme to do its job -High km: low affinity to do its job
• Competitive: The inhibitor has a similar structure to the substrate binding to the original site
• Uncompetitive: The inhibitor does not to the original site
• Non-competitive: can be removed from the substrate by increasing the substrate concentration

Common Types of Reversible Inhibition

• Competitive inhibition: the inhibitor is structurally similar to the substrate and binds in the active site, preventing the actual substrate from biding
• Uncompetitive inhibition: the inhibitor binds only to the ES Complex
• Noncompetitive inhibition: the inhibitor binds either the enzyme or enzyme-substrate complex

Metabolic Strategies

• The generation of energy from food occurs in three stages
• Large molecules in the food stream are broken down into smaller molecules to undergo digestion
• Small molecules undergo processing into key molecules of metabolism, notably the formation of Acetyl CoA
• ATP is produced from the the acetyl component of Acetyl CoA

Basic Principles of Metabolism in Cells

• Molecules degraded or synthesized into a series of pathways
• ATP is the energy currency
• ATP can be formed by the carbon fuel oxidation
• Metabolic pathways are regulated
• A limited number of reaction types are found to occur in metabolic pathways
• Oxidation Reduction: electron transfer
• Group Transfer: transfer of a functional group from one molecule to another
• Hydrolytic: Cleavage of bonds by the additon of water
• Isomerization: Rearrangment of atoms for isometric arrangement
• Ligation: required cleavage
• Redox, refers to election transfer

ATP and Hydrolysis

• Hydrolysis is highly exergonic because the triphosphate unit contains two bonds that are unstable
• AG depends on many factors, but is approximately -50kjmol
• Occurs by means other than hydrolysis/redox reactions

Isomerization

• Citrate to isocitrate is the most well known and studied

Ligations

• Using free energy from ATP cleavage to form new bonds
• Forming bonds by using free energy from ATP cleavage
• A carbon bond is lengthened and pyruvate is formed into oxalcetate
• Energy derived from the fuels are covered into energy through the use of Atp.

Atp acts as a donor

acts as free energy for the use of motion, active and biosynthetic reactions for it requires high amounts of energy Has a higher phosphoryl for glycerol 3-phosphate The energy makes the having phosphoryl:

• reosionance stabilization
• electrostatic repulsion
• increases in entrocy
• stablization by hydration

Oxidation of Carbon Fuels

• Most important to a source for activity
• App for biological activity
• Amount is limited
• must consistantly recycles to provide energy Capture energy of oxidation as ATP Aerboic fuel molecules, atoms to oxidize to C02 to create water
• Fats and oils (Most)

Carbohydrates

• Ketone, alkynes
• atleast 2 hydroxyl groups
• or substances, that yield such substance by hydrolysis Monosaccharidases has a single sugar and a single unit
• oligosacchrides have more than one chain

Forms of Fructoses

• Pyranose to furanose, are named because of resemblamnce of a pyran carbon ring
• Can react by CU32 the anomer forms pass in equilibrium and through a chain of forms and agents. The sugars that react with the oxidizing agents are called reducing sugars, those with out are non reduced
• Glycosidic

Monosaccharides:

• Glycosid bond to form Alcholols and Animes

Lipids

• Cellulose to Homopolymers Glycoproteins, more weight that protein in structure Mucins : proteins is attached by N. acetylgalactosamine

Oligosaccharides

• Contained in bloods: blood groups follow specific pattern: O-all or most blood contains O-types while people with AB types have more enzymes, A-types has glycosyltransferase.

Viral Receptors:

• Virus recoginizes saild acids and binds with the cells.
• Infection happens when infection spreads through the neuraminidase
• causes Glycosid bond by slicing a viral protain freeing the virus.

Protein Glycosylation

O-links happens because of protein glycoslation

Glycosyltransferases

Monosaccharides substrates for glycosyltransferases are activated by the attachment of uridine diphosphate: Glycolisis- uses potent fuel on all forms throughout life

Transporters

• The movement of glucose is facilitated

Glycolysis

• Process :molecule of glucose is degraded for 3. carbons, and results product is pruvate Atp- is consumed to a molecule o glyceralhyde for processing

preparatory phase

A process to break it down and transfer it. energy for Glycolysis: Energy conversed by 2 adp, 2 nadh Payoff: 2 pyruvate at end

Regulation Process

• hexokinase uses active glucose by phosophalating and by humans creates 4 hexokinases from the body that contain reactions. also, reversible
• The hexo I, II, III inhibited at all sides.

Allosteric Regulation

A form of allosteric which means the action is in motion Activation is at ratio of 55 The low Atp or Amp ratio:

• Actrivation from insluan Fructose -2-6 INsulan Repressor: high A,T/Mp ration
• Citrate
• Glucuong

Glucagon signals liver to produced:

• Glucose and releases
• To not over consumed Decreased by pancreas by the blood, releases the actibity
• Insulan promotes, causes, signals to use glucose.

Fructose: reversible and from concentration

• Triosphosphateisomerase: catalization.

Fates of GLucose

• Carbon atoms from C1, C2, C3 are used.

Phosphorylation

Transfer from bisophophoglerate to And Posphohlerate reversible reaction, which is transferable example to tranerring phophate to adp of gdp'

Conversion

reversible shift the reaction is reversible and removal by a molecule through a water process, and pyruvate catalysis,

Regulations pyruvate

insulin releases the action and enzyme works at level

• Glucogon act at the non muscle section. P - high ratio and activation high
• Inhibit and and adp Net Equation for Glucosilysis: 2 to the product are given.

2+4+4+->

Overview of heme synthesis and degradation, enzymes, and metabolism. Heme is made in the liver and erythroblasts (red blood cell precursors). It consists of protoporphyrin IX, iron (Fe), and four nitrogen atoms. Iron is inserted into the protoporphyrin ring by an enzyme.