Biochem

CENTRAL DOGMA - Prelearning

Nucleic Acid – composed of DNA & RNA

DNA

Forms inherited genetic material

Genes code for protein, determine physical traits

Doesn’t direct protein synthesis itself, uses RNA as intermediate

DNA transcribed into RNA to serve as template for protein translation

DNA transcripted into RNA → RNA translated to protein

RNA (detailed later)

DNA Structure – Nucleotides, Double Helix, Base Pair Bonding, Phosphodiester Bonds

Nucleotides

3 components: nitrogenous bases, 5-C sugar, phosphate group

2 types of nitrogenous bases: purines, pyrimidines

Purines = 2 ring base = guanine, adenine

Pyrimidines = 1 ring base = thymine, cytosine

Double Helix

Sugar phosphate backbone maximizes efficiency of base-pairing

3 forces needed to stabilize structure

H-bonds b/w base pairs

Base stacking

Parallel to each other

Expels water from b/w them (hydrophobic effects)

Base Pair Bonding

Held together by H-bonds (A-T, G-C)

A always pairs with T and G always pairs with C because these are the only combinations that allow for hydrogen bonding to occur

A-C cannot form H-bonds, neither can G-T

Chargaff’s Rule - # of purines = # of pyrimidines

Necessary to maintain shape of double helix, which requires there to be one purine and one pyrimidine in each base pair

Shape of DNA helix would change if we had purine-purine, pyrimidine-pyrimidine interaction

If two purines paired there would be four 'rings' and they would distort the structure of the DNA molecule. Two pyrimidines would not easily bond as their single ring structures would not normally span the distance between the two strands

Phosphodiester Bonds

Sugar-phosphate backbone

Nucleotides join together

Covalent phosphodiester bond

Neg charge b/w P groups w/in sugar-phosphate backbone

Adjacent neg charges repel each other, destabilize helix

Positively charged Mg2+ ions help stabilize neg charges (ionic interactions)

Use 5’ and 3’ to denote direction of strand

Summary:

The following bonds and forces contribute to the stability of the DNA helix:

Phosphodiester bond (sugar-phosphate backbone)

Ionic interactions

H-bonds b/w complementary base pairs

Hydrophobic interactions (base stacking)

DNA Condensation – Nucleosomes, Chromatin, Chromosomes

Nucleosomes

Structural unit for packaging DNA

Composed of 147 base pairs wrapped around histone core

Octamer of H2A, H2B, H3, H4

H1 Linker protein

Chromatin

Complex of DNA + tightly bound protein

Heterochromatin – densely packed

Euchromatin – dispersed

Found when cell is transcriptionally active

DNA must be exposed for genes to be transcripted

Chromosomes

Most condensed form of DNA

23 pairs (46 total)

1 copy of each from each parent (2n; diploid)

Maternal/paternal pair = homologous chromosomes (homologs)

Haploid cells

1 copy of each chromosome

Sperm, oocytes (mature egg)

Autosomal

1-22

Form homologous pairs (1 maternal, 1 paternal copy)

Sex Chromosomes

Determine biological sex

Non-homologous

Female = XX, Male = XY

Genes

Functional units of heredity

Segment of DNA w/ instructions to make particular protein

Exon – coding sequence; EXpressed; included in RNA transcript, translated into protein

Intron – non-coding sequence; INTerfering; not included in RNA

Removed via splicing after transcription

Non-Coding DNA

98\.5% of genome doesn’t encode protein, instead regulates gene expression

Promoter & enhancer regions – bind transcription factors

Binding sites for factors that organize chromatin structures

Non-coding regulator RNA – eg. MiRNA

Mobile genetic elements (“transposons”) not well understood

Gene regulation and chromatin organization

RNA

RNA Structure

Polymer made of nucleotides link by phosphodiester bond (like DNA)

Chemically different from DNA

Ribose sugar vs. Deoxyribose

Uracil (U) base instead of thymine (T)

Structurally different

Single stranded (can fold into various shape) vs. double-stranded helix of DNA

Types of RNA – Coding

mRNA

Messenger RNA = transcript of DNA transcribed into RNA as template for protein translation

Directly codes for proteins

Initially pre-mRNA then undergoes processing into mature mRNA

Types of RNA – Non-Coding

Sequences of DNA transcribed into RNA, not translated to proteins

Serve as enzymatic, structural, or regulatory components of cell processes

snRNA

Small nuclear RNA; Functions in spliceosome (needed to removing introns, splicing, from pre-mRNA)

Associated w/ protein subunits to form small nuclear ribonucleoproteins (snRNPs) which form core of spliceosome

rRNA

Ribosomal RNA; Needed for basic structure of ribosome complex

Catalyzes peptide bond b/w AAs

tRNA

Transfer RNA; carry correct AA to polypeptide chain

Unique cloverleaf shape

Anticodon:

3 consecutive nucleotides that pair with complementary codon in mRNA

Amino acid binding site:

Short, single-stranded region at 3’ end of tRNA

Binds AA that corresponds to anticodon on tRNA

Wobble Hypothesis

64 possible combos of nucleotides into 3 nucleotide codon, but only 20 AAs

Demonstrates redundancy of genetic code

More than 1 possible tRNA for many AAs

some tRNA molecules can base-pair w/ more than one codon

Some tRNA only require accurate base pairing of first 2 positions of codon and can tolerate mismatch (aka wobble) in 3rd position

Explains why so many alternative codons for an AA differ only in 3rd nucleotide

Micro RNA (miRNA)

Regulates gene expression via post-transcriptional silencing

Blocks/prevents translation of specific mRNAs; promote degradation

Small interfering RNA (siRNA)

Reduce gene expression

Direct degradation of specific mRNA

Long non-coding RNA (lncRNA)

Regulate gene expression

Increase or decrease transcription

Involved in X-chromosome activation

Types of RNA

Name

Function

mRNA

Messenger RNA

Codes for proteins

rRNA

Ribosomal RNA

Important constituents of ribosomes. Catalyzes protein synthesis

tRNA

Transfer RNA

Adaptor between mRNA and amino acids

snRNA

Small nuclear RNA

Splicing of pre-mRNA

miRNA

Micro RNA

Regulate gene expression: block translation of specific mRNA & promote its degradation

siRNA

Small interfering RNA

Regulate gene expression: direct specific mRNA degradation

lncRNA

Long non-coding RNA

Regulate gene expression: can increase or decrease transcription

CENTRAL DOGMA – In Class

Transcription

Transcription: process of synthesizing RNA molecule from DNA template to dictate synthesis of a protein

Occurs in cell nucleus

Transcriptional Unit

Promotor region – contains consensus sequence

Coding region – DNA transcribed into mRNA

Terminator region – specifies end of transcription; tells us when to stop

Anything before TSS is upstream, anything after is downstream

Template Strand

Strand of DNA that is transcribed into RNA; used to code

Anti-sense strand

Non-template strand – complimentary partner to template strand; not used in coding

Sense-strand

RNA Polymerase

Main enzyme for transcription; moves along DNA

Unwinds DNA helix before active site

Catalyzes new phosphodiester bond on newly-forming strand of RNA\*

Works in 5’ --> 3’ direction

Makes more mistakes than DNA polymerase

OK since only making proteins; AA sequence can make up for it

Mistake is in protein, not genome like DNA’s would be

Steps of Transcription

Initiation

Initiation factors help RNA polymerase recognize where to start

Prokaryotes: only one, Sigma Factor

Eukaryotes: many! TFII (transcription factor 2) is a general transcription factor

TFII binds consensus sequence in promotor region

TATA box (consensus sequence); located upstream from TSS

TFIID is the specific TFII that binds the consensus sequence (TATA)

Within it is the TATA binding protein (TBP)

Other transcription factors join

RNA Polymerase II joins

Transcription initiation complex completes; transcription begins

Summary: First part of transcription is to form the transcription initiation complex (which is composed of TFIID, other TFIIs, and RNA polymerase)\*

Regulation

Repressor proteins bind upstream silencers (aka negative regulatory elements)

Inhibit gene transcription

Transcriptional activator proteins bind upstream enhancers (aka positive regulatory elements)

Increase transcription rate by attracting RNA Polym II

If there was a mutation in either of these, transcription would speed up or slow down, giving us more or less protein in the end, influencing cell function

Ex. of how mutations don’t need to be in genes to influence amount of protein made

Elongation

TFII released once RNA Polym begins transcribing DNA

TFs then available to initiate another round of transcription

RNA moves downstream, transcribes codon region

Elongation factors needed; reduce likelihood of RNA Polym dissociating from DNA\*

Eukaryotes require:

Chromatin remodeling complexes to help RNA navigate chromatin structure

Histone chaperones disassemble and reassemble nucleosomes as RNA Polym passes through

Supercoils generated as RNA Polym moves along

DNA topoisomerase removes supercoiling by breaking phosphodiester bond

Allows two sections of helix to rate freely, relieves tension

Phospho bond reforms as DNA topo leaves

*know that you need RNA Polymerase and DNA Topoisomerase for Elongation*

Processing (specific to Eukaryotes) and Termination

preMRNA transcript processed in 3 ways:


1. Capping 5’ end

Modified guanine nucleotide added to 5’ end of transcribed preMRNA\*

Occurs early

Facilitates export of mRNA out of nucleus; involved in translation


2. Splicing

Introns and exons transcribed into RNA

Introns removed by RNA splicing

Performed by spliceosomes

Require snRNA (small nuclear RNA) and proteins complexed into snRNPs (small nuclear ribonucleoprotein)

SnRNP = snRNA + proteins

Allows ONE gene to produce variety of diff. Proteins

Mutation can occur; too much or too little intron could be removed → folding impacted → structure impacted


3. Polyadenylation of 3’ end

3’ end of mRNA is specified by signals encoded in DNA → transcribed into RNA → bind to proteins that facilitate cleavage of mRNA from RNA Polym

Once termination signals are transcribed, mRNA is cleaved from RNA Polym

Once mRNA is cleaved, poly A tail added (\~200 nucleotides added)

Poly A tail protects mRNA from degradation; facilitates export from nucleus

First termination, then Poly A binding proteins then bind poly-A tail to 3’ end

Once complete, transcript is mRNA

Prokaryotes

Steps are same, except:

No processing required (no 5’ cap, splicing, or poly-A tail)

No export from nucleus (translation beings ASAP)

Transcript is polycistronic

Multiple genes w/in transcript; transcript codes for more than one protein

In Eukaryotes, one transcript for one gene

Translation

After processing & termination, mature mRNA exported from nucleus

Once in cytosol, translated into protein

MRNA decoded in sets of 3 nucleotides called codons (ex. AUG is a codon); correct sequence depends on correct reading frame

In Eukaryotes, AUG is first sequence

In Prokaryotes, it is Shine Dalgarno sequence (ribosome binding site)

Additional recognition sequence needed for ribosome binding

If a nucleotide was accidentally inserted into middle of a gene, it would mess up subsequent codon, end up w/ wrong AAs

Molecules needed for translation:

MRNA transcript

“charged” tRNA (bound to correct AAs)

Ribosomes (large and small subunit

Preparing tRNA

Cell makes a variety; each corresponds to one of 20 AAs

Aminoacyl-tRNA synthetase catalyzes attachment of correct AA to tRNA

Basic enzyme name – each AA has it’s own specific tRNA synthetase name (ex. Tryptophanyl tRNA synthetase)

Once tRNA is bound to codon in RNA, tRNA is charged (need aminoacyl tRNA synthetase)

Ribosomes

Where protein synthesis is performed

Maintains correct reading frame; ensures accuracy of codon-anticodon interaction

Composed of ribosomal proteins + ribosomal RNA (rRNA)

Large subunit – involved in catalyzing new peptide bond (has E, P, A binding site)

Small subunit – mRNA binding site w/in small unit

*know we need: mRNA transcript, charged tRNA (w/ aminoacyl tRNA synthetase), and ribosomes (rRNA + protein)*

Steps of Translation

Initiation

AUG, methionine, is first codon translated on mRNA; start codon

\*all newly made proteins begin w/ methionine as their first AA @ N-terminus

Met-tRNAi - Forms initiator tRNA-methionine complex

Loaded into small ribosomal subunit P site w/ initiation factors (eIFs)

Small ribosomal subunit binds to 5’ end of mRNA

modified guanine cap recognized 5’ end

Small ribosome moves along mRNA from 5’ to 3’ scanning for AUG

Requires ATP hydrolysis; expensive energy-wise

Once AUG found, initiation factors dissociate

large ribosomal subunit assembles; complete ribosome complex

\*Met-tRNAi binds P site of small subunit → small s.u. binds to 5’ cap → scans mRNA until AUG → large ribosome s.u. added → complex complete

Elongation

tRNA binding

Charged tRNA binds to A-site of ribosome complex

Peptide bond formation

Carboxyl end of chain released from tRNA at P site → joins AA linked to tRNA at A site

Catalyzed by peptidyl transferase in large ribosomal s.u.

Large subunit translocation

Large r.s.u. moves relative to mRNA held by small s.u.

Two tRNAs are shifted to E and P sites

Keeps shifting; what was the A site becomes the P site

Small subunit translocation

Small s.u. shifts by 3 nucleotides

TRNA in E site is ejected

Cycle repeated for new incoming aminoacyl-tRNA

Elongation factors help; coupled w/ GTP Hydrolysis

\*charged tRNA binds w/ A site → peptide bond formed (peptidyl transferase) → large subunit translocation → small subunit translocation

Termination

End of translation marked by STOP codon

UAA, UAG, UGA

Not recognized by tRNA; do not specify an AA

Release factors bind to ribosomes w/ STOP codon at A site

Peptidyl transferase catalyzes addition of H20 molecule rather than AA

Frees carboxyl end, releases polypeptide

Ribosome releases mRNA, dissociates into large and small subunits

Subunits recycled for new round of protein synthesis

mRNA is now a polypeptide w/in cytosol

Polysomes

Synthesis of proteins occurs on polyribosomes (group of ribosomes)

Multiple initiations take place on each mRNA molecule being translated

As soon as preceding ribosome moves out of way, new ribosome complex is formed

Helps speed up rate of protein synthesis

Post Translational

Protein folded into specific 3D shape

Structure dictates function; proper folding important

May be modified in ER (proteins, sugars added)

Sent to proper cell location (cytosol, nucleus, etc.)

Regulation of Protein Synthesis

Cell can regulate amount of protein available to cell by regulating:

Amount of transcription that occurs

Stability of mRNA transcript

Location of protein

Destruction of protein (later)

Transcription Regulation

Histones

Highly dynamic; regulated by many nuclear proteins

Chromatin remodeling complexes – reposition nucleosomes on DNA to expose or obscure gene regulatory elements (ex. Promoters)

Chromatin writer complexes – carry out histone modifications (methylation, acetylation, phosphorylation)

Histone acetylation – opens chromatin & increases transcription

Performed by histone acetyltransferases (HAT)

Euchromatin (active)

Deacetylation reverses changes; promotes chromatin condensation

Heterochromatin (inactive)

Histone methylation – promotes transcriptional activation/repression (DNA and RNA)

DNA methylation results in transcriptional silencing

MRNA stability

Longer mRNA in cytosol, more protein made via translation

Non-coders (miRNA, siRNA) promote destruction of mRNA transcript, OR

Specific proteins bind to mRNA, prevent degradation, resulting in more protein synthesis

Ex. Transferrin – brings iron into cell

If low iron levels, mRNA is stable, transferrin receptor made

If high iron levels, mRNA degrades, no receptor made

We decrease amount of transferrin protein by increasing degradation

Targeting cell location

Cell can limit protein to particular location

If needed for intracellular use, translated on free ribosomes in cytosol

If needed for nucleus, mitochondria, or peroxisomes:

Specific AA signal targets protein for correct location

If needed for lysosomes, ER, cell membrane or secretion:

Directed to rough ER co-translational transfer

Translation begins on free ribosome in cytosol

Signal peptide sequence translated, binds signal recognition particle (SRP)

Binding SPR stops translation, directs ribosome to RER, binds to SRP receptor

Translation restarts, polypeptide moves into lumen of RER, signal peptide sequence removed

If protein needs to be in cell membrane, will contain stop transfer sequence

Translation is paused when it comes in contact with translocator

Translocator discharges polypeptide into phospholipid bilayer of ER

Translation resumes until polypeptide is complete

PROTEINS + ENZYMES 1 (Pre-Learning)

Protein Classification

Shape

Fibrous Protein

Globular Protein

Long and rod-shaped

Compact & spherical

Generally has structural function

Provides strength

Generally has dynamic function

Eg. Enzymes to catalyze reactions

Eg. Carrier proteins

Often insoluble in water

Often soluble in water

Eg. Keratin, Collagen

Eg. Enzymes, albumin, hemoglobin

Composition

Simple – composed only of AAs

Conjugated – composed of protein portion + non-protein portion

Protein portion – contains only AAs

Non-protein portion – prosthetic group

Conjugated protein w/o prosthetic group is called “apoprotein”

Protein Structure

Primary

Polypeptide chain

Linear sequence of AAs

AAs held together by peptide bond

Secondary

Repeating backbone formed by H-bonds b/w carboxyl and amino groups

Alpha helix

Each COOH group H-bonds w/ amino group 4 AAs away

Rigid, rod-like structures

Beta pleated sheet

2+ polypeptide lined up side-by-side

Held together by H-bonds between distant carboxyl and amino groups

Tip points in C terminal direction

Super-secondary structure are a combo of alpha helices and/or beta-pleated sheet

Tertiary

3D structure created by side chain interactions

H-bonds

Salt bridges

Disulfide bridges

important in extracellular proteins

Strong bonds: help protect protein from denaturation during changes in blood pH or salt concentrations

Insulin held together by disulfide bonds

Hydrophobic interactions

Quaternary

Association of all multiple polypeptide subunits

Functional protein

Ex. Hemoglobin

4 subunits: 2 alpha, 2 Beta

Final 3D shape of protein dictates its function

Dimer – protein composed of 2 subunits

Oligomer – protein composed of several subunits (ex. Hemoglobin)

Multimer –protein composed of many subunits

Protomer – repeating structural unit w/in multimeric protein

Hemoglobin has a pair of αβ protomers

Protein Folding

After translation, proteins fold into secondary, tertiary, and quaternary shapes

Chaperones – proteins that help others

Fold into correct shape

Get to correct cell locations

Heat Shock Protein (HSP) is a common one

Binds and stabilizes portions of protein not yet folded

Eventually released via ATP hydrolysis

Refold proteins partially unfolded due to stress

PROTEINS + ENZYMES 1 (in class)

Protein Denaturation

Protein Denaturation

Occurs when bonds holding protein together are disrupted

Loss of protein structure (disruption in folding/shape)

Disrupts secondary and tertiary structures

Bonds w/in proteins can be disrupted, and proteins denatured, by using:

Strong acids or bases, or reducing agents:

These add or remove hydrogens

Organic solvents, detergents:

Disrupt hydrophobic, polar and charged interactions

Salts:

Disrupt polar and charged interactions

Heavy metal ions:

Ex: Mercury (Hg+2) and Lead (Pb+2)

Based on their positive charge, they can bind to negative amino acids with acidic R groups

Denature proteins by disrupting salt bridges

Have high affinity for thiol/sulfhydryl (SH) groups

Alters shape of protein

Basis of “lead poisoning”

Enzymes

Protein catalysts that speed up rxns and remain unchanged\* by the rxn

Speed up by lowering Activation Energy Ea of the rxn, without changing:

Standard free energy (ΔG)

Equilibrium

Activation Energy (Ea)

Minimal amount of energy to make/break bonds necessary for rxn to occur

Also defined as amount of energy needed to reach transition state (TS)

TS = highest energy config. Formed when changing from reactants → products

Transient and not isolated

If enzyme present, aka catalyzed rxn, Ea is lowered; if no enzyme, Ea is higher.

Specificity

Enzymes = highly specific

Only substrate of correct size and shape can enter into active site

Active site – special cleft in enzymes, forms by precise quaternary structure of protein

AAs in active site participate in substrate binding and catalysis

Once inside site, substrate binds to enzyme forming enzyme-substrate (ES) complex

Induced fit model – binding of substrate induces conformational change in shape of E

Mechanisms

Induced fit b/w substrate and enzyme allows for:

Electrostatic interactions to form b/w them

Correct positioning of catalytic groups

Catalytic groups speed up rxns in two main ways:\*

Acid-base catalysis

Covalent catalysis

Acid-Base Effects

Side chains of certain AAs add/remove H+’s by acting as acids or bases, making substrate more reactive

Ex. Histidine

Functions as both an acid or base (pKa of 6)

Often a catalytic acid w/in an enzyme to assist w/ acid/base effect

Adding an acid catalyst speeds up rxn, adding base catalyst speeds up rxn, adding both an acid and a base speed it up even more

*catalytic AAs can either add or remove H+’s to speed up rxns*

Covalent Catalysis

Nucleophilic side group in enzyme active site forms temporary covalent bond w/ substrate

Ex. Asp and Glu (R-COO-)

Ser (R-OH) and Cys (R-SH)

Only weakly nucleophilic; enhanced by presence of other AAs that can remove H

Bond forms temporarily, then goes back to normal

Role of Cofactors and Coenzymes

Cofactors and coenzymes help enzymes speed up rxns in three main ways:

Help position substrate on active site of enzyme

Stabilize negative charges on substrate or TS to make easier for nucleophilic attack to occur

Can accept/donate e- in redox rxns

Cofactors – metal cations, Mg2+, Zn2+

Mg2+ neutralizes negative charges on ATP

Helps position ATP on enzyme active site

Coenzymes – derived from vitamins

B2 – FADH2 – redox rxns

B3 – NADH – redox rxns

Can accept or donate electrons in redox rxns

B6 – PLP – transamination, decarboxylation

B9 – 1C transfers (eg. Methylation)

Cofactors and coenzymes only assist the enzyme, they don’t carry out their functions

Effect of temperatures or pH

Temperature

Optimal temperature for enzyme = temperature of organism

pH

Changing pH changes protonation state of enzyme (E) and/or substrate (S)

Disrupts:

H bonds

If H removed, no bond can be formed

If H added, bond might form where it is not supposed to

Electrostatic interactions

Will disrupt salt bridges/ionic bonds

Adding H can turn COO- into COOH, Removing H can turn NH3+ into NH2

Lysosome – intracellular enzymatic degradation for many molecules; breaks down proteins

Active at pH 4.5

Regulation

Enzyme activity controlled in four main ways

Genetic

Enzyme transcription can be induced/repressed based on cell’s needs

Induced in times of need

Enhancer regions of DNA

Repressed when in excess

Ex. Silencer regions of DNA

Other examples: transcription regulation (enhancer/silencer regions), modifying histones (chromatin remodeling and rewriting complexes, histone acetylation, histone methylation), mRNA stability

Slow process; in times of excess we increase transcription. In times of abundance, we decrease

Ex. Regular consumption of high sugar food → high insulin → increased transcription of genes for glucokinase, PFK-1, and pyruvate kinase → increased translation → higher amount of glucokinase, PFK-1, and pyruvate kinase in cytosol → more efficient conversion of glucose to pyruvate

Covalent modification

Involves altering the structure of enzyme (“proenzyme”) by making/breaking covalent bonds

Two types:

Reversible

Addition/removal of group to enzyme that causes it to convert to active or inactive form

Phosphate group commonly does this

Can activate or inactivate the enzyme

Methyl and acetyl groups also common

Glycogen metabolism

Main enzyme in glycogenesis is de-activated by phosphorylation while main enzyme in glycogenolysis is activated by phosphorylation

Catalyzed by protein kinase A (PKA)

Prevents both pathways from running @ same time

By simply adding P group, we activate one and inhibit other

Know that adding/removing P group is an example of reversible covalent modification and can either activate or inhibit an enzyme\*

Irreversible

Cleavage of peptide bonds in proenzymes or zymogens to form active enzyme

Ensures enzyme isn’t used until in correct location and needed

Can be turned into an active enzyme by being turned on and off

Allosteric Modification

Binding to allosteric site changes conformation and activity of enzyme

Changes binding affinity of substrate @ active site

Have more than one subunit

Allosteric site on one, active site on another

Binding of effector molecule to allosteric enzyme can either:

Increase binding of substrate to enzyme

Effector = activator

Decrease binding of substrate to enzyme

Effector = inhibitor

Consider glycolytic enzyme PFK-1

PFK1 is inhibited allosterically by high levels of ATP

When there is a lot of ATP in cell, PFK1 will decrease its affinity for F6-P

It means we have a lot of energy, so we are going to slow down how quickly we go through glycolysis b/c we don’t need this additional energy

PFK1 is activated allosterically by high levels of AMP

High AMP increases affinity because AMP is a sign the cell is low in energy. We want to go through glycolysis faster so we have more energy

Binding AMP to PFK-1 increases it’s activity

Compartmentalization via membrane-bound organelles

Allows for regulation by:

Separation of enzymes from opposing pathways into diff compartments, and selective transportation of substrates

Ex. Two opposing metabolic pathways compartmentalized in diff. Areas of the cell are fatty acid synthesis (cytosol) and beta oxidation (mitochondria)

Creation of unique microenvironments

Lysosomal enzymes function at pH around 4.5, while most others function at pH 7

\*know the categories; be able to pick out an example. Ex. What ATP binds to this enzyme, it’s affinity for this substrate increases. (Answer: allosteric); OR. If we increased transcription of this gene, what type of regulation is that? Genetic

Which of the following is an example of enzyme regulation via reversible covalent modification?

a. Phosphorylation of glycogen synthase inhibits glycogenesis

b. A high carbohydrate diet increases the transcription of the gene for pyruvate kinase

c. Cleavage of a peptide bond in a proenzyme to form an active enzyme

d. Binding of AMP to phosphofructokinase-1 increases its activity

ENZYME KINETICS

Michelis-Menten Kinetics

Determine if an enzyme is physiologically useful based on its:

Maximum rate

Max speed that enzyme can convert a substrate into its product

Affinity for substrate (or inhibitor or coenzyme…)

Basically gives an idea of how likely the enzyme is to bind a substrate and will do so even if concentration is low

If affinity is low, you will need high concentration of your substrate before enzyme can bind

Can be applied to certain rxns:

First order reactions

Concentration of a single substrate is directly proportional to rate of reaction

Straight line: \[s\] is proportional to rate; substrate concentration is proportional to the rate of the rxn

Many physiological rxns involve more than a single substrate

A single substrate can control the rate even when there is more than one substrate involved

Ex. You have enzyme that has two substrates, A and B

One A and many Bs

Only one B enzyme would be able to bind to A substrate

If you added more As to the solution, you will increase rxn rate

If you added more Bs, nothing would happen because we already have enough

This is a case where if you have two substrates, only one of them will affect the rate of enzyme

Called a pseudo first order rxn

Only one substrate affected the rate, but really, there are two substrates

In a fumarate to malate reaction, it is a hydration reaction

The substrate ignored in M & M kinetics is water (because adding more water would do nothing to the rxn)

In a acetylcholine to acetate and choline rxn, it is a hydrolysis rxn (using water to break molecule)

The substrate ignored again is water; acetylcholine is the single substrate that will affect the rate

Zero order rxn

Rate stops increasing even though substrate concentration continues to increase

Enzyme is already saturated so adding more substrate will not increase rxn rate

Equation

Vo: initial rate of rxn

S is high, P is low, therefore no reverse rxn takes place

Km: indication of how well the enzymes bind given a molecule

Small Km = high affinity; large Km = low affinity

Graphing S against rxn rate determines:

Vmax (max. Rate)

Km (enzyme affinity)

Lineweaver-Burk found it was not so easy to get info out of graph with that equation

Created reciprocal of M & M equation that creates a straight-line graph, easier to extrapolate

Y intercept = 1/Vmax

Extrapolated x-intercept = -1/Km

On a graph, the LOWEST intercept line is going to be the highest Vmax (because the Y axis is 1/Vmax)

On a graph, the furthest from the XY intercept line is the lowest Km value, therefore greatest affinity

Enzymes Part 2 - Kinetics

Application of Km and Vmax: Glucokinase and Hexokinase

Hexokinase (most tissue) contrasts w/ glucokinase (liver) by the following:

Hex has lower Vmax, lower Km, greater affinity for glucose

Turned off by high concentrations of glucose 6-P

Both HexoK and GlucoK catalyze Glucose → Glucose 6-P

Glycolysis breaks down glucose

Glycogenesis stores glucose

Glucokinase higher Km tells us it has lower affinity for glucose, compared to hexokinase

Hexokinase is active during fasting

Greater affinity, can convert even small amounts of glucose into energy

Glucokinase only becomes active after a high-carb meal

Lower affinity, needs a lot of glucose to be activated

Main role in glycogenesis because you want to store this excess

Liver

Where GK is found

Nutrients absorbed from intestines go here first; GK converts excess glucose from a meal to glycogen

Other tissues cant convert excess glucose to glycogen because Glucose 6-P inhibits Hexokinase (product inhibition)

Hexokinase only works until you have made enough G6-P to serve body’s energy needs

HK feeds into glycolysis; explains low Km because even though you have little glucose, HK binds to it because it has a greater affinity

Makes sense that GK has a higher Km because we do not want it storing glucose unless we have already satisfied our energy needs

HK is inhibited by G6-P because it is mediating glycolysis; once you’ve made enough G6-P to fulfill you energy needs, HK turns off and GK turns on to store as glycogen for later use. Once glucose levels fall, GK turns off because it has a low affinity for glucose

Vmax

High Vmax = high capacity to turn substrate into product

Allows GK to phosphorylate glucose to glucose 6-P after a meal to store as glycogen

Adding P to glucose means it gets trapped in the cell

Km

Helps determine enzyme efficiency

How well it binds (affinity) and how quick it converts to product

Kcat/Km

Kcat measures speed of product formation once ES has been made

Km measures binding affinity of E and S to make ES

E + S