Biotech Exam 2

L10

proteins functions

• most of cell’s dry mass

• versatile functions

  • storage

  • transport

  • movement - motor

  • structure

  • signal transduction - receptors

  • growth/development - hormones

  • immune protection - defense

  • catalysis - enzyme

• structure determines function

categories of amino acid side chains

• acidic (negative charge) polar

• basic (positive charge) polar

• uncharged polar

• nonpolar

polypeptide chain directionality

orientation of a protein chain, running from an N-terminus (amino end) to a C-terminus (carboxyl end)

protein domains

• in large proteins

• compact modular units that fold more or less independently

• 40-350 continuous amino acids

• diff domains associated w diff functions

• ex: cytochrome b562, NAD binding domain of lactate dehydrogenase, SH2 domain, Src protein formed from 3 domains

complex assemblies

• dimer/homodimer → a protein complex containing of 2 identical subunits

• tetramer → hemoblobin; 4 heme groups

• sacromere → myosin and actin

protein denaturation

• when a protein is converted into a randomly coiled peptide without its normal activity

• primary structure is not active

• due to heating, chemical denaturants, oxidizing or reducing agents

• may lose biological function

native protein

a protein in its folded and active form

protein biosynthesis

translation of nucleotide sequences into amino acid sequences

• same basics across all kingdoms of life

• ribosomes are responsible for this process

codon

set of 3-letter combinations of nucleotides, which corresponds to a specific amino acid or stop signal

transfer RNA (tRNA)

adaptor molecule in protein synthesis; contains an amino-acid recognition site as well as a template-recognition site (anticodon)

• each tRNA sequence can be arranged in a cloverleaf pattern

• activated amino acid is attached to a hydroyl group of the A residue in amino-acid-attachment site of CCA sequence at 3’ end

• anticodon loop - makes 3 bases that constitute the anticodon accessible for interaction w an mRNA codon

activation of amino acid

1. formation of an adenylated amino acid

2. attachment of amino acid to corresponding tRNA

aminoacyl-tRNA synthetase

enzyme that catalyzes the aa activation reaction and linkage of aa to tRNA

• activates tRNA by coupling it w its respective tRNA molecule

L10 NOT DONE YET

L13

irreversible inhibition

covalent binding of inhibitor to the enzyme

• cannot be broken

• forms stable complex

reversible inhiibition

inhibitor forms noncovalent bonds w enzyme

• can dissociate

• inhibitor can be unbound

suicide inhibition

an irreversible form of enzyme inhibition

• inhibitor binds to active site of enzyme and forms an irreversible complex thru a covalent bond during normal catalysis reaction

• ex: aspirin, penicillin, fluorouracil

fluorouracil (5-FU)

anticancer drug that acts as suicide inhibitor of thymidylate synthase

• converted in vivo into flouorodeoxyuridylate (F-dUMP)

• F-dUMP irreversibly inhibits thymidylate synthase after acting as a normal substrate through part of the catalytic cycle

used to treat breast cancer, colon or rectal cancer, pancreatic cancer, and stomach cancer

types of reversible inhibition

• competitive inhibition

• uncompetitive inhibition

• non-competitive inhibition

competitive inhibition

the inhibitor competes w the substrate for binding to the enzyme; binds to:

• active site

• anywhere else on the enzyme

ex: statins, ACEi

allosteric competitive inhibitor

binds to allosteric site, changes the conformation of the active site and the substrate can no longer bind there

statins as competitive inhibitors

competitively inhibit HMG-CoA reductase enzyme

• cholesterol-lowering drugs

uncompetitive inhibition

inhibitor binds only to the enzyme-substrate complex

• can prevent catalysis rather than substrate binding

ex: camptothecin (topoisomerase inhibitor that binds to topoisomerase I and DNA complex - anti-cancer drug)

non-competitive inhibition

the inhibitor binds either the enzyme or enzyme-substrate complex

• can prevent catalysis rather than substrate binding

ex: doxycycline - collagenase inhibitor → preserves collagen in tissues; used in periodontal disease, osteoarthritis, RA

types of non-competitive inhibitors

• pure non-competitive inhibitors

• mixed non-competitive inhibitors

pure non-competitive inhibitors

bind equally well to the enzyme w or without substrate bond

mixed non-competitive inhibitors

bind preferentially to either the free enzyme or the enzyme-substrate complex

enzyme kinetics

V = rate of enzyme reaction

Vmax = max rate of a reaction

Km = substrate conc at which the reaction velocity is half of the max

competitive inhibitor enzyme kinetics

• inc in apparent value of Km

• Vmax remains unchanged

• can be overcome by a sufficiently high [S]

non-competitive inhibitor enzyme kinetics

• lowers conc of functional enzyme

• Vmax is dec

• Km is unchanged

• cannot be overcome by inc substrate conc

uncompetitive inhibitor enzyme kinetics

• inhibitor binds to ES (enzyme substrate) to form ESI (enzyme substrate inhibition complex), depleting ES

• Vmax is dec

• Km is dec

• dec in free enzyme correspond to an enzyme w greater affinity for its substrate

competitive inhibitor as drugs

a higher conc of the inhibitor is req for competitive inhibition when substrate conc is high

• equally potent

• equally selective

noncompetitive inhibitor as drugs

unaffected by high substrate conc

• equally potent

• equally selective

majority of molecular targets of FDA approved drugs

human and pathogen proteins

pic - drug targets span a variety of protein classes

antibiotics

compounds produced by bacteria and fungi which are capable of killing or inhibiting competing microbial species

penicillin

• one of the most widely used antibiotics in the world

• penicillium mold naturally produces the antibiotic penicillin

• against many bacterial infections caused by staphylococci and streptococci

• treatment of diphtheria, gonorrhea, pneumonia, syphilis, and others

• estimated to have saved > 200 million lives

penicillin mechanism of action

• interferes w synthesis of cell walls of reproducing bacteria

  • defective walls cause bacterial cells to burst

  • human cells are not affected bc they do not have cell walls

• irreversibly inhibits transpeptidase

  • transpeptidase → enzyme that catalyzes the last step in bacterial cell-wall biosynthesis

• suicide inhibitor

aspirin

• extracts form the bark and leaves of the willow tree

• active compound → salicylic acid

• acetylsalicylic acid (aspirin) made by neutralizing salicylic acid w a base and acetyl chloride

aspirin mechanism of action

• irreversible inhibitor of COX (cyclooxygenase) enzymatic activity of prostaglandin H2 synthase (PGH2)

• prostaglandins promote the inflammatory response (swelling, pain, fever)

• COX inhibitors blunt this response

protease inhibitors

block protease activity

ex: HIV-1 protease, thrombin, ACE

ACEi (angiotensin converting enzyme inhibitor)

competitively block the ACE enzyme, which prevents to production of angiotensin II

• relax veins and arteries to lower bp

• protect kidney function in patients w diabetes or mild kidney disease

angiotensin II

• hormone that circulates in blood

• main role is to constrict blood vessels

• can cause high bp

• present in abnormally high quantities in many cardiovascular diseases

ACE

• zinc metalloproteases

• hydrolyze peptides

• convert inactive angiotensin I to angiotensin II by removing a dipeptide

thrombin

converts fibrinogen to fibrin to create a blood clot

• serine protease, final enzyme of blood coagulation cascade

blood clotting cascade

• enzymatic cascade - series of zymogen activations → rapid response

• activated form of one clotting factor catalyzes the activation (enzyme) of the next precursor

• signal is amplified at each step

• a very small amt of initial factors suffice to trigger the cascade → rapid response to trauma

anticoagulant drugs

meds that reduce blood clot formation; prevent stroke, myocardial infarction, and pulmonary embolism in patients w inc clotting risk

DTIs (direct thrombin inhibitors)

anticoagulant drugs; serine protease inhibitor

• univalent → bind only to active site

  • ex: argatroban, melagatran

• bivalent → bind to both active site and regulatory sites

  • ex: hirudin, bivalirudin

warfarin

anticoagulant drug; vitamin K antagonist

• inhibits enzymes req to regenerate the active form of vit K

• vitamin K: essential for the synthesis of prothrombin and several other clotting factors

HIV protease

cleaves multidomain viral proteins into their active forms

• aspartyl protease

HIV protease inhibitors

inhibit HIV protease

• ex: indinavir, retrovir

• blocking this process completely prevents this virus from being infectious

• in combo w inhibitors of other key enzymes have dramatically reduced deaths to AIDs

indinavir

one of the 1st HIV protease inhibitor

• designed to resemble a peptide substrate of the enzyme

• OH group interacts w 2 aspartate residues of the active site of HIV proteases but generally absent in cellular aspartyl proteases → specificity

paxlovid

SARS-CoV-2 protease inhibitor; antiviral drug combo that treats COVID-19

• bioavailable orally

• prevents transmission

• reduce hospitalization rate and mortality

• ex: nirmatrelvir and ritonavir

nirmatrelvir

3C-like (3CL) protease inhibitor

• 3CL → protease that SARS-CoV-2, the virus that causes COVID-19, uses to make more copies of itself

ritonavir

strong cytochrome p450 (CYP3A4) inhibitor and pharmacokinetic boosting agent

kinases

transfer a gamma-phosphate group from ATP to serine, threonine, or tyrosine residues of a protein

• > 518 protein kinases on the human genome (1.7% of human genes)

• kinases phosphorylate more than 30% of the cellular proteins

• classified by amino acid substrate preference

  • serine-threonine kinases

  • tyrosine kinases

  • dual kinases (phosphorylate serine, threonine, or tyrosine residues)

phosphatatases

transfer the phosphate from a phosphoprotein to a water molecule

major kinase classes and immunoreceptor signaling

timeline of approval of key protein kinases inhibitors

some kinase inhibitors are used to treat cancers

imatinib (gleevec)

• breakthrough in cancer treatment

• highly specific inhibitor of bcl:abl kinase

  • forms due to chromosomal translocation and overexpression of bcr:abl fusion genes

• used to treat CML

  • occupies the ATP-binding site

  • targets tumor cells specifically, minimizing side effects

• 83.3% rate of 10-year overall survival

• inhibitor of several tyrosine kinases

  • c-kit, a protein tyrosine kinase in GI stromal tumors

  • PI3K in solid tumors

  • serine/threonine kinase BRAF in melanoma

  • the receptor tyrosine kinase EGFR in lung cancer

  • serine/threonine kinase mTOR in renal tumors

FLT3 inhibitors for the treatment of AML

[2017] midostaurin → first approved FLT3 inhibitor in US

[2018] gilteritinib → received FDA approval in US

[2019] quizartinib → recieved approval in Jaban

[2023] quizartinib → received approval in US

L14

maintaining homeostasis

req metabolic regulation that coordinates the use of nutrient pools

• diet

  • proteins

    • amino acids

    • body protein

    • amino acid pool

    • gluconeogenesis

      • urine

  • carbohydrates

    • glucose

    • glycogenesis

    • lipogenesis

    • glucose pool

      • brain metabolism

  • lipids

    • free fatty acids + glycerol

    • fat stores

    • free fatty acid pool

      • metabolism in most tissues

overall protein digestion

• dietary proteins → degraded to amino acids which are absorbed by the intestine and transported in blood

• cellular proteins are degraded to amino acids bc of damage, misfolding, or changing metabolic demands

• excess amino acids → cannot be stored or directly excreted, so they must be used for protein synthesis or degraded to ammonia

protein homeostasis

• rate of synthesis = rate of degradation

• required to maintain a physiological concentration req for life

essential amino acids

in humans must be obtained through diet; cannot be synthesized by body; 9 amino acids

protein digestion

• begins in stomach where acidic environment denatures proteins into random coils by pepsin

• partly digested proteins move from stomach to beginning of small intestine (duodenum) → stimulating secretion of NaHCO3 and proteolytic enzymes from pancreas

• aminopeptidases in plasma membrane of intestinal cells enhance digestion

• free amino acids, dipeptides, and tripeptides are transported into intestinal cells

• at least 7 diff transporters specific to a diff group of amino acids

• absorbed amino acids are released into blood by a number of Na+ - amino acid antiporters

pepsin

the primary proteolytic enzyme of the stomach; max active at pH 2

protein turnover

degradation and resynthesis of proteins

• takes place constantly in cells

• essential for removing short-lived or damaged proteins

half-lives of proteins range over several orders of magnitude

• ornithine decarboxylase (catalyzes synthesis of polyamines) - 11 minutes

• hemoglobin - life of RBC

• lens protein crystallin - life of organism

ubiquitin

a small (76 aa) protein that tags proteins for destruction

• present in all eukaryotic cells

• highly conserved

• has extended carboxyl terminus, which is activated and linked to proteins targeted for destruction

regulation of protein turnover

• ubiquitin attaches by its carboxyl-terminal Gly residue to the epsilon-amino groups of 1+ Lys residues on the target protein

  • req ATP hydrolysis

  • forms an isopeptide bond bc epsilon- rather than alpha-amino groups are targeted

attachment of ubiquitin to a protein

• ubiquitin-activating enzyme (E1) → adenylates ubiquitin and transfers it to a sulfhydryl group of a Cys residue of E1

  • req ATP

• ubiquitin-conjugating enzyme (E2) → transfers ubiquitin to one of its own sulfhydryl groups

• ubiquitin-protein ligase (E3) - transfers ubiquitin from E2 to an epsilon-amino group on the target protein

  • brings E2 and the target protein together

  • ubiquitin be transferred directly or be passed to a Cys residue of E3 first

multiple ubiquitin molecules are typically added to a single protein substrate

• E3 can remain bound to target protein and generate a chain of ubiquitin molecules

• E3 can dissociate after the first ubiquitin addition, and a chain can be extended by another E2/E3 pair

• ubiquitin can be added onto any of the 7 Lys or the N-terminus of the previous ubiquitin

• a chain of 4+ ubiquitin molecules linked via Lys 48 is an especially effective signal for protein degradation

E3 ubiquitin ligases provide protein target specificity

in humans

• there are 2 E1 ligase genes

• there are 30-50 E2 ligase genes

• there are over 600 E3 ligase genes

in tetraubiquitin chain, 4 ubiquitin molecules are linked

• linked by isopeptide bonds

• the epsilon-amino group of a Lys residue of one ubiquitin is linked to the terminal carboxylate of another

• this unit is the primary signal for degradation when linked to a target protein

importance of E3 proteins to cell function

• proteins not degraded bc of a defective E3 may accumulate, causing a disease of protein aggregation

• angelman syndrome → severe neurological disorder of an unusually happy disposition, cognitive disability, absence of speech, uncoordinated movement, and hyperactvitity

  • caused by defect in a member of E3 family

• overexpression of E3 ligase (UBE3A) is strongly linked to certain forms of autism

• inappropriate protein turnover can lead to cancer

  • many tumor suppressors, including BRCA1, are E3 ligases

additional roles of ubiquitination

regulates proteins involved in

• DNA repair

• chromatin remodeling

• innate immunity

• membrane trafficking

• autophagy

proteasome

• 26S proteasome - large, ATP-driven protease complex that digests ubiquitinated proteins

• complex of 2 components

  • one 20S catalytic unit arranged as a barrel

  • two 19S regulatory units that control access to interior of the 20S catalytic subunit

function of 19S regulatory unit

• contain ubiquitin receptors that bind specifically to polyubiquitin chains

• use ATP to unfold polyubiquitinated chains and direct them into the catalytic core

• contain an isopeptidase that cleaves off intact ubiquitin molecules so that they can be reused

• 6 ATPases of the AAA+ class

  • AAA+ class → class of chaperone-like ATPases associated w assembly, operation, and disassembly of protein complexes

20S catalytic unit

• barrel-shaped

• made up of 28 homologous subunits

  • alpha-type and beta-type are arranged in 4 rings of 7 subunits each

  • some of beta-type subunits include protease active sites at the amino termini

• active sites in the beta subunits

  • chymotrypsin-like → cleaves after large hydrophobic amino acids

  • trypsin-like → cleaves after basic amino acids

  • caspase-like → cleaves after acidic amino acids

• all active sites employ an N-terminal Thr residue

  • the OH group of Thr residue attacks the carbonyl groups of peptide bonds, forming acyl-enzyme intermediates

  • substrates are degraded in a processive manner without intermediate release

  • substrates are reduced to peptides ranging from 7-9 residues before release

proteasome and other proteases generate free amino acids

• ubiquitinated proteins are processed to peptide fragments

• ubiquitin is removed and recycled prior to protein degradation

• peptide fragments are further digested to yield free amino acids, which can be used for biosynthetic reactions, most notably protein synthesis

  • alternatively, the amino group can be removed and processed to urea and the C skeleton can be used to synthesize carbohydrate or fats or used directly as a fuel for cellular respiration

processes regulated by protein degradation

many biological processes are controlled, at least in part, by protein degradation via the ubiquitin-proteasome pathway

bortezomib (velcade)

dipeptidyl boronic acid inhibitor of the proteasome; used as a therapy for multiple myeloma

HT1171

suicide inhibitor of the proteasome of M. tuberculosis; has no effect on human proteasomes

amino acid degradation

• amino acids not needed as building blocks are degraded to compounds able to enter the metabolic mainstream

• major site of degradation → liver

• muscles also readily degrade the branched-chain amino acids (Leu, Ile, Val)

amino acid degradation steps

1. amino group is removed & remaining carbon skeleton is metabolized to a glycolytic intermediate or to acetyl CoA

2. alpha-amino groups transferred to alpha-ketoglutarate, yielding glutamate

3. glutamate is oxidatively deaminated in the liver to yield ammonium ion

4. ammonium ion is converted to urea, which is excreted

aminotransferases

catalyze the transfer of an alpha-amino group from an alpha-amino acid to an alpha-ketoacid; req coenzyme pyridoxal phosphate (PLP) - derivative of pyridoxine (vit B6)

• reactions are reversible and can be used to synthesize amino acids from alpha-ketoacids

• presence of alanine and aspartate aminotransferases in the blood is an indication of liver damage

  • liver damage due to:

    • viral hepatitis

    • long-term excessive alcohol consumption

    • reaction to drugs

  • liver cell membranes are damaged and aminotransferases leak into blood

aspartate aminotransferases (AST)

catalyzes the transfer of amino group of aspartate to alpha-ketoglutarate

alanine aminotransferase (ALT)

catalyzes the transfer of amino group of alanine to alpha-ketoglutarate

pyridoxal phosphate (PLP) enzymes

• at the alpha-carbon of amino acids, PLP-dependent enzymes catalyze

  • decarboxylations

  • deaminations

  • racemizations

  • aldol cleavages

• at the beta-carbon and gamma-carbon of amino acids, PLP-dependent enzymes catalyze

  • elimination

  • replacement reaction

glutamate dehydrogenase

mitochondrial enzyme that converts N atom in glutamate to a free ammonia ion by oxidative deamination

• liver-specific enzyme

• can use NAD+ or NADP+

• proceeds by dehydrogenation of the C-N bond, followed by hydrolysis of the ketimine

• allosterically inhibited by GTP and stimulated by ADP in mammals

serine and threonine can be directly deaminated

• serine dehydratase and threonine dehyrdatase directly deaminate their respective amino acids

  • PLP is the prosthetic group

• no transfer of the alpha-amino group to alpha-ketoglutarate is req

• dehydration precedes deamination

  • serine → pyruvate + NH4+

  • threonine → alpha-ketobutyrate + NH4+

N from amino acids is cleared through the urea cycle in the liver

peripheral tissues transport N to the liver as glutamine

• N is transported to liver as glutamine thru glutamine synthetase to avoid high levels of circulating ammonia

• N of glutamine are eliminated by incorporation into urea in the liver

glutamine synthetase

catalyzes the synthesis of glutamine from glutamate and NH4+

muscle cells transport N to liver as alanine by glucose-alanine cycle

• during prolonged exercise and fasting, muscle used branched-chain amino acids as fuel

• the N removed is transferred (through glutamate) to alanine, which is released into the blood stream

• in liver, alanine is taken up and converted into pyruvate for the subsequent synthesis of glucose

urea cycle overview

• 2 N atoms enter the cycle and leave as urea

• CO2 is simultaneously eliminated as it is hydrated to bicarbonate, which then enters the cycle

• urea cycle, gluconeogenesis, and transamination of oxaloacetate are linked by fumarate and aspartate

urea cycle steps

1. ammonia + bicarbonate → carbamoyl phosphate via carbamoyl phosphate synthetase I in the mitochondria

   1. carbamoyl phosphate synthetase I req allosteric regulator N-acetylglutamate for activity

2. carbamoyl phosphate + ornithine → citrulline via ornithine transcarbamoylase in the mitochondria

3. citrulline is transported into the cytoplasm

4. citrulline + aspartate (donor of second N in urea) → argininosuccinate via argininosuccinate synthetase in the cytoplasm

5. argininosuccinate → arginine + fumarate via argninosuccinase

   1. fumarate is hydrated to malate, which is oxidized to oxaloacetate

   2. oxaloacetate converted to glucose by gluconeogenesis OR transaminated to aspartate for another round of urea synthesis

6. arginine is hydrolyzed → ornithine + urea

   1. ornithine is transported back into the mitochondria

   2. urea is excreted

carbamyl phosphate synthetase I

catalyzes the coupling of ammonia with bicarbonate to form carbamoyl phosphate

• mammals have 2 isozymes

• req 2 molecules of ATP, making the rxn essentially irreversible

• req the allosteric regulator N-acetylglutamate for activity

• inhibited by acetylation and stimulated by deacetylation

N-acetylglutamate synthase

catalyzes the synthesis of N-acetylglutamate

• activated when amino acids are readily available

ornithine transcarbamoylase

catalyzes the transfer of the carbamoyl group of carbamoyl phosphate to ornithine, forming citrulline

• occurs in the mitochondria