Neostigmine
What does this DRUG inhibit?
Acetylcholinesterase
IRREVERSIBLE ENZYME INHIBITOR
Organo-arsenicals
Pyruvate Dehydrogenase
IRREVERSIBLE ENZYME INHIBITOR
D-cycloserine
Alanine Racemase
IRREVERSIBLE ENZYME INHIBITOR
Azaserine
Formylglycinamide Ribonucleotide AMINOTRANSFERASE
IRREVERSIBLE ENZYME INHIBITOR
4-hydroxy-androtenedione
AROMATASE
IRREVERSIBLE ENZYME INHIBITOR
Chloramphenicol
Peptidyl transferase
IRREVERSIBLE ENZYME INHIBITOR
5-fluorouracil
Thymidylate Synthase
IRREVERSIBLE ENZYME INHIBITOR
Disulfram
Aldehyde Dehydrogenase
IRREVERSIBLE ENZYME INHIBITOR
What is a Suicide Substrate?
and what are Examples?
The inhibitor is UNREACTIVE until the enzyme
tries to USE IT as a substrate.
covalent -> permenant inhibition
Ex.
Penicillin / Physotigmine / 5-fluorouracil
ASPIRIN is NOT a suicide substrate
it will REACT even W/O an active site
How is Acetylcholine broken down and why?
It is TOXIC if released in EXCESS
ACETYLCHOLINESTERASE = AChE
converts ACh to a less toxic choline
Acetylcholinesterase
- *SERINE HYDROLASE**
- inactivates Acetylcholine*
contains a key serine residue in the active site that reacts with a phosphorus-group of certain NERVE TOXINS
irreverisibly inactivates AChE
–> DEATH
Inhibitors of AChE = Parathion / Sarin / Dursban
Sarin
Covalent modification:
Irreversibly inhibits ACETYLCHOLINESERASE
- *key serine** in AChE’s active site –> attacks Sarin’s P-group
- irreverisbly inactivates AChE*
How do Heavy Metals cause TOXICITY?
Mercury / Lead / Silver
Iron + Copper
Heavy metals –> form tight bonds w/ SULFHYDRYL GROUPS
that are needed for catalytic activity or structural reasons
Cys / Disulfide bridges / Lipoate / CoA
Irreversibly inhibit catalysis function
&
Distort the STRUCTURE of the enzyme
Non-Covalent Regulation
Allosterism
&
Effectors
Types of COVALENT regulation
- *Reversible**
- *Phosphorylation / Energy Charge / Acetylation**
Irreversible
Proteolysis / Glycosylation
methylation / fatty acids
Types of COVALENT Modifications
Glycosylation
Methylation
Reversible Phosphorylation
Acylation (esp Acetylation)
FA’s
Proteolysis
What does GlycoSylation ADD? and TO WHAT?
Type of COVALENT Modifications
irreversible attachment of 1 or more SUGARs by glycosylases
in the golgi / ER
- *Bulky / Polar / Solvated**
- *RECOGNITION ELEMENTS**
Sugars-Oxygen –> SERINE residues
sugars-Nitrogen –> ASPARAGINE residues
What is the FUNCTION of GLYCOSYLATION?
Type of COVALENT Modifications
add sugars to:
Directing Enzyme** to its **Proper Cellular Location
for proper Folding/activation & release from cell
Also important for:
Cell-cell ADHESION
RECOGNITION by the immune system
PROTECTING proteins from attack
What does METHYLATION ADD? and to WHAT?
Type of COVALENT Modifications
METHYL group to the terminal amino group on a LYSINE
may be REVERSIBLE, demethylases can remove methyl group
SAM = S-AdoMet
the MAJOR donor of in vivo methyl groups
Folate donates -Ch2 groups
Biotin carries the -COOH group
What is the FUNCTION of METHYLATION?
Type of COVALENT Modifications
methyl group –> Lysine
Affects the ELECTRICAL PROPERTIES on the amino groups
important for HISTONES –> in their role for gene expression
What is SAM?
what are its functions
S-AdenosylMethionine,
major donor for METHYLATION
Synthesis of:
epinephrine / phosphtidylcholine / creatine
Methylation of nucleic acid bases
Methylation of LYS RESIDUES IN HISTONES
What does Reversible Phosphorylation ADD, & TO WHAT?
Type of COVALENT Modifications
Kinase adds Phosphate groups using ATP
Ser / Thr / Tyr using the -OH sidechain
Phos-STT
Introduces a Charged / Bulky group that alters:
- *Conformation** / state of aggreagetion
- *Blocks sites**
- *Attract/repel small molecues**
Appears in many
Energy-Producing** & **Energy-Consuming Pathways
What is the FUNCTION of Reversible Phosphorylation?
Type of COVALENT Modifications
phosphate -> STT (ser / thr / tyr) on -OH
ENERGY PATHWAYS
both Consuming & Producing
Kinase ADDS P w/ ATP
PhosphoTASES remove phosphate
PhosphoRYLASE do NOT use ATP -> use inorganic phosphate
Kinase Function
Catalyze the ADDITION of Phosphate groups
using ATP
in reversible phosphorylation
PhosphaTASE Function
TASE = REMOVES phosphate
in reversible phosphorylation
Phosphorylase Function
LASE = LACK ATP
DOES NOT USE ATP, uses Inorganic Phosphate
in reversible phosphorylation
Examples of Phosphorylation REGULATION
Type of COVALENT Modifications
- *Metabolic Enzymes**
- *Glycogen synthase** / Acetyl CoA carbodylase / PDH
Cytoskeletal Proteins
desmin / vimentin / caldesmon
- *Nuclear Proteins**
- *CREB** / progestrone / RNA polymerase
- *Membrane** Proteins
- *Insulin receptor** / EGFR / B-adrenergic receptor
What does Acylation ADD? and to What?
Attaches FATTY ACIDS –> ESTER
range from acetyl groups (2C) –> Long chain FA’s
BULKY & Electrically NEUTRAL Group
as ANCHORS
What is the FUNCTION of ACYLATION?
Type of COVALENT Modifications
Neutral / Bulky Fatty Acid
Interfere with:
Protein-Protein Assocaition
Conformational Change / binding of amall molecules
LONG-CHAIN FA = hydroPHOBIC –>
can Hold Proteins in Membrane
an maybe direct the polypeptide to proper cell location
Types of ACYLATION use to help proteins to associate w/ MEMBRANEs
Type of COVALENT Modifications
- *GPI Anchors**
- -> external leaflet = RAFT
N-Terminal Mystic Acid Tail
SATURATED –> RAFT
C-terminol Sterols
Saturated -> RAFT
Cys Acylation
Isoprenylation
Lipids that go on RAFTS
Acylation
SATURATED lipid tails, RIGID / TIGHT
think Saturated = no kinks, full of double bonds
GPI-Anchored
Sterol-Linked
Palmitoylated intracellular
Lipids that go on NON-raft regions
UN-saturated Tails
kinds = FLUIDITY
Prenylated
Palmitoyl
Caveolae
SMALL CAVES associated with LIPID RAFTS
high in CHOLESTEROL & SPHINGOLIPIDS
involved in:
transmembrane transport & Signal transduction
What does ACETYLATION ADD? and to what?
Type of COVALENT regulation
- *Acetylases** add 2-Carbon FA
- -> END of the side chain of Lys** _(_amide linkage)**
- DEacetylases REVERSE this process*
A type of Acylation, as common as phosphorylation
is often REVERSIBLE, like phosphoylation
What is the FUNCTION of ACETYLATION?
Type of COVALENT regulation
2-Carbon FA –> LYS side chain, amide linkage
LOSES the + Charge on LYS
affects:
substrate binding / assocation
CHANGE IN ENZYME ACTIVITY
activated or inhibited (MDH in TCA / SDH in TCA)
Metabolic State
Cell Regulation
Acetylation & Metabolic State
Acetyl groups -> LYS
generated by the breakdown of FA’s / AA’s / CARBS
High Levels of Acetyl Groups = HIGH ENERGY
Acetylation & CELL REGULATION
2C-FA -> LYS
- *acetyltransferase / deacetylases** often found in
- *nucleus / mito / cito**
Metabolism
ENZYMES
- *Cytoskeleton**
- *a-tubulin**, is aceytlated -> regulates STABILITY of microtubules
- *GENE EXPRESSION**
- *histones** -> various tx factors + coregulators
Why do Cell proteins NOT last FOREVER?
Damaged / MISfolded
Need to be REMOVED as a part of the cell cycle
or to CONTROL a metabolic pathway
or as a part of Programmed Cell Death = Apoptosis
for TURNOVER** = **AA’s can be RECYCLED
What is TURNOVER?
the RECYCLING of AA’s from proteins
Protein Breakdown / Re-Conversion to Short Peptides / AA’s
2 main pathways:
Vacuolar / Cytoplasmic
cysteine proteases = caspases
2 Main Pathways of AA Recycling = TURNOVER
VACUOLAR
done by lysosomes / endosomes / ER
Cytoplasmic
done by ubiquitin / Proteasomes
- Minor pathway* = caspases = cysteine proteases
- *programmed cell death**
What is PROTEOLYSIS?
AMIDE BOND CLEAVAGE
a type of Cytoplasmic Turnover = Inactivate protein/enzyme
issue is balancing & the costs of new synthesis
Generally irreversible modification
unlike phosphorylation & acetylation
Can ALSO lead to ACTIVATION
inactive precursor -> cleaved to produce an ACTIVE anzyme
in digestive / blood clotting / insul
What are LYSOSOMES?
Type of VACUOLAR Turnover:
Hydrolytic Sacs
ATP-Driven Proton PUMP –> ACIDIFIES INSIDE
Acid Hydrolases for:
proteins / carbs / nucleic acids / lipids
active ONLY @ ACID PH
Lysosomal Functions
Large molecules brought in by
vesicles that merge w/ lyososme membrane
METABOLITE TRANSPORTERS
expel the resulting monomers
primary route for recycling / turnover of:
AA / Sugars / Simple Lipids
Lysosomal Diseases
Tay-Sachs** / **Pompe Disease
Defects in 1+ hydrolases –> “choke” the lysosome
w/ undigested large molecules –> disease
Buildup of undigested carbs –> stress the cell
Cytoplasmic TURNOVER
“Mark & Chew”
1st stage = UBIQUINATION
marks the proteins for degradation, w/ ubiquitin (protein)
2nd = PROTEOSOMAL action
- *proteasome** –> hydrolyzes PEPTIDE bonds
- -> releases AA’s & short peptides
Proteasome Inhibitors as DRUGS
Proteasomes regulate cell cycle by degrading key proteins used for rapid synthesis / signal proteins
Disrupting proteasomes -> HALT Growth / proliferation
Applications of inhibition of proteolytic Activity:
- *Anticancer / Antiviral** Drugs
- *Anti-Inflammatory / Anti Tuberculosis**
Ishema / Stroke damage alleviation
