18: Enzyme Categories and their role in human health

which enzymes can break peptide bonds and how do they do it?

proteases

hydrolysis of peptide bonds (add water to break bond)

which reaction is thermodynamically favorable and kinetically extremely slow? what is the halflife of this reaction without an enzyme?

hydrolysis of peptide bonds

10-1000 years

explain the hydrolysis of peptide bonds

water added to break bonds

OH added to cut carbonyl (making carboxylic acid)

H added to N creating amine

proteolytic enzyme that participates in the breakdown of proteins in the digestive system

chymotrypsin

chymotrypsin cleaves peptide bonds selectively on the carboxyl terminal side of large hydroPHOBIC residues such as

phenylalanine

methionine

tryptophan

tyrosine

what makes chymotrypsin a serine protease?

there is a serine in its active site where the peptide will bind

how does chymotrypsin (serine protease) bind to its protein substrate?

covalent and transiently (only for a short amount of time)

which two phosphorus based agents modify reactive serine residues?

why is this toxic?

• Malathion and Sarin

• serine residue on enzyme is needed to break apart gut proteins (chymotrypsin)

which proteases can cleave peptides at other places besides serine residues?

• cysteine proteases

• aspartyl proteases

• metalloproteases

which protease relies on a cys residue to be activated by a His residue to play the role of the nucleophile that attacks the peptide bond

cysteine protease

which protease uses a pair of Asp residues to allow a water molecule to attach the peptide bond (hydrolysis)?

aspartyl protease

which proteases use a bound metal ion (typically zinc) to activate a water molecule to act as a nucleophile to attach the peptide carbonyl group?

metalloprotease

which enzyme is essential for the following industries?

• dairy

• food

• detergent

• leather

• waste

• pest

• contact lens cleaning

• prion protein degradation

• wool bio polishing

• animal feed

• therapeutics

• silk degumming

• peptide synthesis

• biofilm degradation

PROTEASE

the human carbonic anhydrase II is bound to

zinc and carbon dioxide

what is the end product to aerobic metabolism?

what do carbonic anhydrases do to the end product of aerobic metabolism?

• carbon dioxide

• carbonic anhydrase converts CO2 into HCO3- and H+ in red blood cells

• it can also do the REVERSE and dehydrate bicarbonate ion (HCO3-) in the blood to form CO2 for exhalation

does carbonic anhydrase only convert CO2 into H+ and HCO3- (bicarbonate) ?

NO

it can also dehydrate the bicarbonate and turn it into H+ and CO2

Carbonic Anhydrase has an essential role in regulating

BLOOD pH

instead of serine like many proteases, what is bound to carbonic anhydrase that allows it to catalyze its reaction?

Zinc

bacterial enzymes that degrade viral DNA

restriction endonucleases (restriction enzymes)

what can restriction endonucleases be used for?

• eliminate viral infections

• recognize base sequences called RECOGNITION SEQUENCES in DNA molecules (cognate DNA)

• characterize and manipulate DNA

which type of restriction enzymes cleave DNA within their recognition sequences?

type 2 restriction enzymes

restriction enzymes catalyze the hydrolysis of

what is the end result

phosphodiester backbone of DNA

• between 3” oxygen atom and phosphorous atom

• DNA strands with a free 3’ hydroxyl group and 5’ phosphoryl group at the cleavage site

what is attached to the active site of the restriction enzymes that is required for hydrolysis

what does this cofactor do?

magnesium or other divalent cations (2+)

forms bridge between the enzyme and DNA substrate

cofactor for

• chymoprotase

• carbonic anhydrase

• restrcition enzymes

• serine

• Zn2+

• Mg2+ or other 2+

what forms the bridge between the DNA substrate and restriction enzyme

Mg2+ or other divalent cation

can restriction enzymes cut any DNA you want it to?

what is a caution to have when dealing with restriction enzymes?

NO there must be a recognition sequence

you want it to cut DNA at recognition sequences of VIRAL DNA not your OWN DNA

what prevents restriction enzymes from cutting human host DNA that is found within the recognition sequence?

how is this done?

• host DNA within the recognition sequence is methylated at their adenosine bases

• methylation is done by DNA methylase

what is the restriction modification system?

restriction enzyme + DNA methylase

hydrolyzing only Viral DNA without attacking the methylated adenosine bases of the host DNA

how are restriction enzymes utilized in the following:

• DNA profiling (DNA fingerprinting): detects ___________ _______ responsible for disease and determine whether food is free of _____________ ____________

• Genetic engineering

  • Medicine: mass production of human _______, __________ ____________, ____________ ____________, antihemophilic factor, vaccines, and other drugs

• Industry:

  • mass production of _________ by growing the transformed organism in bioreactors using ________, then purifying the ________ (enzymes for laundry detergents or cheese)

• Agriculture: create ________ __________ crops and organisms (resistant to insecticides)

• bacterial strains pathogenic bacteria

• insulin, growth hormones, monoclonal antibodies

molecular motor proteins

myosins

how do myosins drive molecular motion?

they use thermodynamically favorable reactions

• use conformational changes to harness ATP hydrolysis to mechanical work

myosin coverts __________________ energy —> ___________ energy

chemical —→ mechanical

Myosins have elongated structures with

• __________ ATPase domains that carry out _______ ________ at one end

• extended ___-_______ structures that promote _________ FORMATION

• ancillary associate proteins termed ________ _________

• globular ATP hydrolysis

• a-helical DIMER

• light chains

what must happen for ATP to be hydrolyzed by myosin

what does ATP hydrolysis allow the myosin to do?

conformational change

move along actin filamints

the actin filament has a ______________ structure

each actin monomer contains a bound _________ ( ______ or ________)

polymeric

nucleotide ATP or ADP

Myosin Movement along Actin SEQ

1. what happens once ATP binds to myosin?

2. what happens when ATP is reversibly hydrolyzed?

3. once ATP is hydrolyzed into ADP it stays bound to ________ allowing myosin to bind to actin once again

4. The release of _____ reorients the lever arm (a-helix) and concomitant motion of actin relative to myosin

5. ________ is released

1. Myosin head is released from actin

2. the a-helix lever arm of myosin reorients (conformation change)

3. actin

4. Pi

5. ADP

SEQ Myosin + Actin

1. ATP binds to myosin releasing it from actin

2. ATP hydrolysis allows conformational change movement of a-helix tail

3. ADP + Pi allow myosin to reattach to actin

4. Pi leaves allowing for conformational change and myosin to move along actin

5. ADP leaves

at what steps does the lever arm (a-helix) of myosin undergo conformational change?

once ATP is hydrolyzed and once Pi is released

How can you control Enzyme Activity?

1. _____________ control by regulatory molecules

2. __________ enzymes that catalyze the same reactions but exhibit different _________ and ____________ properties

3. reversible ___________ modifications

4. ____________ proteolytic cleavage to yield an active enzyme product

5. controlling the _____ of enzyme present

1. allosteric (bind to another location on the enzyme to prevent substrate binding)

2. alternate catalytic regulatory

3. covalent

4. irreversible

5. amount

_________- binding includes many active sites and/or regulatory sites

allosteric

allosteric enzymes usually consist of multiple _________

the binidng of substrate to one active site can alter the __________ of the other active sites in the same molecule through __________ changes

subunits

properties conformational

do allosteric enzymes obey michaelis-menten kinetics?

V= Vmax [S] / Km + [S]

NO

what does the graph of the substrate ([S]) vs Reaction Velocity (Vo) look like?

sigmoidal plot

T vs R state of Allosteric Enzymes:

which is preferred when there is a high amount of substrates? why?

which has low catalytic activity?

which has a higher affinity to bind to substrates?

• R state because the allosteric enzyme is active and ready to bind to substrates

• T

• R

the following describes

• low affinity for substrate

• low catalytic activity

• is favored in the absence of substrate

T state of allosteric enzymes

the following describes

• has higher affinity for substrate

• most active form

• favored upon binding of substrate

R state of allosteric enzyme

What is the allosteric coefficient (L)

[T] / [R]

are only [R] allosteric enzymes present when there is a lot of substrates?

NO both exist and interconvert but [R] is preferred when there are a lot of substrates

the sigmoidal curve for an allosteric enzyme is a composite of two _____________curves

what are the two curves?

Michaelis- Menten

R state curve at high [S]

T state curve at low [S]

_____________push towards the T curve

_____________push towards the R curve

inhibitors

activators

what is a common allosteric regulation method?

feedback inhibition

explain the feedback inhibition of the allosteric regulation

if there is too much product then negative feedback will lower the activity of the first enzyme of the chain reaction of enzymes that led to the product

cells use allosteric feedback regulation to

• slow down production

• conserve energy

• keep a state of balance and homeostasis within the cell

__________ provide a means of regulation specific to distinct tissues and developmental stages

isozymes

enzymes that differ in amino acid sequence that catalyze the same reaction

isozymes

isozymes

• typically display different _________ parameters or respond to different _______ molecules

• encoded by different _________

• may be expressed in a ______-specific or ________ stage- specific pattern

• kinetic regulatory

• genes

• tissue development

catalyzes a step in anaerobic glucose metabolism and glucose synthesis

Lactase Dehydrogenase

humans have two _____________ polypeptide chains for Lactase Dehydrogenase (LDH)

isozymic

what are the two isozymic polypeptide chains for Lactase Dehydrogenase?

what is different about them? what is the same?

1. H= heart muscle

2. M= skeletal muscle

they are both involved in ANAerobic glucose metabolism and synthesis but they differ in their tissue-specificity and genes

can the isozymes for lactase dehydrogenase exist within the same tissues?

where is each mainly found?

YES in the red blood cell, brain, kidney ect.

BUT MAINLY
H = heart

M= muscle

there are two isozymic polypeptide chains for LDH but how many isozymes exist? what are they?

5

1. H4

2. M4

3. H2M2

4. HM3

5. H3M

phosphorylation, dephosphorylation, acetylation, and deacetylation are all ___________________ ____________

are they reversible?

covalent modifications

YES

Kinases transfer a _______-______ group from __________ to a _______ ________ or __________ residue of a protein

• gama phosphate

• ATP

• serine threonine tyrosine

phosphatases transfer the phosphate from a ______________ to a _______ molecule

phosphoprotein water

many folded forms of enzymes are activated by specific ___________ ______________ which is irreversible/reversible

proteolytic cleavage

IRREVERSIBLE

what is the folded inactive enzyme which is only activated through proteolytic cleavage called ?

zymogen

does proteolytic cleavage require ATP or any other energy source

NO

which digestive zymogens in the stomach and pancreas must first be proteolyzed before they can hydrolyze foodstuffs themselves?

what are their activated forms after proteolysis?

pepsinogen—> pepsin

chymotrypsinogen—>chymotrypsin

trypsinogen —→ trypsin

procarboxypeptidase—> carboxypeptidase

Apoptosis is mediated by _________ which are synthesized as ___________

caspases procaspases

Blood clotting is mediated by a cascade of ___________ activators

proteolytic

hormones including _______ are synthesized as inactive precursors which will be activated once they are proteolysed

insulin

__________, the major constituent of skin and bone is derived from __________

collagen procollagen

which systems have proteolytic cleavage to activate enzymes?

• digestive

• apoptosis

• blood clotting

• hormones

• collagen

cleavage of Chymotrypsinogen by _________ initiates the conformational transition into active Chymotrypsin

trypsin

how can you change the amount of enzyme in order to regulate it?

• alter transcription of enzymes gene

• degrade messenger RNA for enzyme

• post-translational changes (protein translocation/degradation)