Proteins and Enzymes Flashcards
What is phenylketonuria?
Faulty enzyme phenylalanine hydroxylase - can’t break down amino acid phenylalanine and it and its toxic derivatives accumulate in the body resulting in braindamage
What is Haemophilia?
Blood clots less easily due to lack of clotting factor
What constituents make up an amino acid?
Hydrogen atom, amino group, carboxyl group, distinctive R group
Name the non polar amino acids
Glycine, Alanine, Valise, Leucine, Isoleucine, Methionine, Phenylalanine, Tryptophan, Proline
Name the polar amino acids
Serine, threonine, cysteine, tyrosine, asparagine, glutamine
Name the acidic (negatively charged) amino acids?
Aspartame, Glutama
Name the acidic (negatively charged) amino acids?
Aspartame, Glutamate
Name the basic (positively charged) amino acids?
Lysine, arginine, histadine
What are the aromatic amino acids?
Phenylalanine, tyrosine, tryptophan
What are the sulphur containing amino acids?
Cysteine, Methionine
Which is an imino acid?
Proline
Amino acids have optical activity, which form are all amino acids found in?
L-isomers
What is the primary structure of a protein, including bonds?
Polypeptide, contains only peptide bonds
What is the secondary structure of a protein?
Spatial arrangements of amino acids in peptide chain, beta pleated sheet or alpha helix, held together by H bonds
What is the tertiary structure of a protein?
3D shape of a protein, held together by H bonds, salt bridges, Van Der Walls forces, Hydrophobic effect, Disulphide bonds
What is the quaternary structure?
Arrangements of different sub units, contains more than 1 polypeptide chain and may include prosthetic groups
What is the name of an amino acid in a polypeptide chain?
A residue
What kind of reaction is a peptide bond formed by?
A condensation reaction, and is a covalent bond, its formed between the carboxyl group of one amino acid and the amino group of the next
Why does a peptide bond have more rigidity and less mobility than a normal covalent bond?
It has double bond characteristics, resonates between 2 forms, a single bond and double bond
What is the structure of alpha helix?
Main chains from inner part of the rod and side chains extend outwards
How is an alpha helix stabilised?
Hydrogen bonds between the amino and carbonyl groups of the main chain, each carboxyl oxygen is H bonded to the amino group 4 residues ahead in the linear sequence
What is the structure of beta pleated sheet?
R groups point outside of the plane of the sheet, the strands can be parallel or anti parallel
How is beta pleated sheet stabilised?
Stabilised by intrachain H bonds, or by interchain H bonds
In tertiary protein structure, what are salt bridges?
Electrostatic interactions between oppositely charged side chains, called an ion pair
In tertiary protein structure what are van der walls forces?
Non covalent forces of attracting between neutral molecules (dipoles), all amino acids form these
In tertiary protein structure what is the hydrophobic effect?
The most effective factor in stabilising protein structure, hydrophobic residues are buried in the core and hydrophilic residues are on the outside
Which 2 residues do disulphide bonds form between?
Between 2 cysteine residues
How do proteins fold?
Proteins attain their final conformation as the lowest energy and most stable shape in seconds, small regions of relatively stable structure form first with the subsequent development of tertiary structure
Protein misfolding an Alzheimer’s disease?
Amyloid precursor protein on plasma membrane is misfolded, cleaved by enzymes, BACE and PS1 to leave a beta Amyloid which is made up of mostly beta pleated sheet which, this accumulates to form beta Amyloid plaques which damage neurons and cause the symptoms associated with the disease
Protein misfolding and Creutzfeldt Jacob disease?
May be inherited or caused by ingested prison protein, contact of normal, soluble PrPc protein (mostly alpha helical) with abnormal PrPSc (mostly Beta pleated sheet) causes the PrPc protein to acquire the abnormal PrPSc structure. Results in loss of neurological function, memory loss, loss of coordination and language ability. Then eventually coma then death
Why are oxygen binding proteins required?
Oxygen has limited solubility in aqueous solutions
What is myoglobin used for?
Oxygen storage mainly in muscle
What is haemoglobin used for?
Oxygen (and CO2) transport in the blood
What is the brief structure of myoglobin?
Globin protein + haem prosthetic group
What is the brief structure of haemoglobin?
4 sub units, each with one haem group
What are the subunits in adult haemoglobin?
2 alpha and 2 beta chains
What is the structure of the ahem molecule?
Fe2+ and a protoporphyrin ring, iron atom coordinates with 6 ligands: 4 N’s from protoporphyrin ring, N from proximal His and an oxygen atom
Binding of O2 in myoglobin?
Non cooperative
What factors affect affinity of myoglobin for O2?
Not pH or CO2 or BPG affects affinity of myoglobin for O2
Which has a greater affinity for O2, myoglobin or haemoglobin?
Myoglobin, myoglobin is almost saturated at O2 concentrations within tissues
What shape is the O2 binding curve of haemoglobin?
Sigmoidal
What is cooperative binding in haemoglobin?
Binding of one O2 molecule makes it easier for the next to bind
What effect does the binding of O2 have on the structure of the Hb subunit?
Binding of O2 pulls Fe2+ into the plane of the harm molecule ring (Proximal His is pulled in), This alters the position of Helix F which then alters the position of the sub units relative to one another rupturing salt bridges and allowing the alpha, beta pairs to slide and rotate, this changes haemoglobin from the tense state to the relaxed state and increases the affinity of other sub units for O2
What is the tense state of Hb?
More salt bridges between sub units, low affinity for O2
What is the relaxed state of Hb?
Fewer salt bridges between sub units, higher affinity for O2
What is the Bohr effect?
Lowering pH decreases the affinity of Hb for O2 (so its released more readily), Bohr shift occurs, ice. The Hb binding curve shifts to the right
Why does lowering pH cause a Bohr shift?
Lowering pH protonates Histidine residues which causes more salt bridges to be formed and stabilises Hb in the tense state (lower affinity for O2, O2 more readily released)
In terms of the Bohr shift when does decreasing pH occur in the blood?
Muscles release lactic acid when respiring anaerobically and produce large amounts of CO2 which also lowers pH when rapidly respiring - allows greater release of O2 when demand is highest
What does BPG do?
BPG is highly negatively charged and binds in a pocket in between the 4 subunits of Hb only in the tense state, so binding of BPG holds more Hb in the tense state so therefore causes a Bohr shift (Affinity for O2 reduced and O2 released more readily to the tissues)
What happens when BPG levels rise?
Release of O2 to the tissues increases
When do BPG levels rise?
BPG is found at high concentrations in all erythrocytes, Raised BPG levels in erythrocytes occur during hypoxia or poor oxygenation of peripheral tissues
What is the relationship between BPG and foetal haemoglobin?
Foetal Hb contains 2 alpha and 2 gamma subunits and binds BPG less effectively than adult Hb, therefore has a higher affinity for O2 than adult Hb and can get O2 from mothers blood
Why is the effects of pH, CO2 and BPG additive?
They interact with Hb at different sites
What are the 2 most common haemoglobinopathies?
Sickle cell anaemia and beta thalassaemia
What is sickle cell anaemia?
Mutation affecting the function of Hb synthesised. Abnormal haemoglobin, at low O2 cells become irreversibly sickle shaped and rupture more easily
What is beta thalassaemia?
Mutation affecting the amount of Hb synthesised, don’t produce sufficient amounts of Hb as cant make beta chains
What is a conservative mutation?
Conservative mutations: Result in an amino acid change. However, the properties of the amino acid remain the same
What is a non conservative mutation?
Mutation resulting in a residue change, involve the replacement of a residue with another with a very different R group
What is the significance of proline residue in a protein?
It is an imino acid and is involved in helix termination
What residue is in the invariant position F8 in haemoglobin and what is its role?
Histidine, Proximal His
What residue is in the invariant position E7 in haemoglobin and what is its role?
Histidine, Distal His
What residue is in the invariant position CD1 in haemoglobin and what is its role?
Phenylalanine, Haem contact
What residue is in the invariant position F4 in haemoglobin and what is its role?
Leucine, Haem contact
What residue is in the invariant position B6 in haemoglobin and what is its role?
Glycine, B and E helices contact
What residue is in the invariant position C2 in haemoglobin and what is its role?
Proline, Helix Termination
What residue is in the invariant position HC2 in haemoglobin and what is its role?
Tyrosine, H bonds between H and F helices
What are collagens?
A family of insoluble fibrous proteins with high tensile strength and are the most abundant protein in the human body, present in most organs, performs a major structural role in a wide range of tissues
What are the 3 main classes of collagens?
1) Fibril forming collagens eg. Types, 1,2,3 (chains/ropes)
2) Network forming collagens eg. Types 4 & 7 (web)
3) Fibril-associated collagens eg. Types 5,9,12
Where does the first step in collagen biosynthesis take place?
In fibroblast cells
Where the second part of collagen biosynthesis take place?
In the extracellular space
What is the simple repeated sequence that makes up much of the primary structure of collagen?
Glycine- Proline - Hydroxyproline (Hyp)
What are the 2 modified amino acids which are common in collagen?
Hyroxyproline (HyP) and Hydroxylysine (HyL)
Which enzymes synthesise the modified amino acids common in collagen and what co factor do they require?
Prolyl and Lysyl Hydroxylases, these enzymes require ascorbic acid (vit. c) as a co factor
What is the role of the 2 modified amino acids in collagen?
HyP is involved in stabilising the molecule through H bonding, HyL is involved in glycosylation (attachment sites for sugar residues) and formation of covalent cross links between collagen chains
What is the secondary structure of collagen?
Each polypeptide form a left handed helix (tighter than an alpha helix and in the opposite direction)
What is tropocollagen?
Triple helix formed from 3 collagen chains which may or may not be identical, the triple helix is a right handed twist making it very stable and Gly residues are packed in the center (small sized R group vital) and HyP and HyL residues on the outside
In collagen synthesis where does assembly of procollagen chains and assembly of triple helix take place?
In fibroblast
In collagen synthesis where does cleavage of propeptides, assembly of collagen into fibrils and formation of covalent cross links take place?
In the extracellular matrix
What are the first steps in the assembly of collagen?
Collagen chains are synthesised with additional amino acids at each end - extension peptides
Assembly begins with disulphide bond formation between C terminal extensions, facilitating triple helix assembly, triple helix (tropocollagen) is then secreted into the ECM
What are the first steps in the assembly of collagen?
Collagen chains are synthesised with additional amino acids at each end - extension peptides
Assembly begins with disulphide bond formation between C terminal extensions, facilitating triple helix assembly, triple helix (tropocollagen) is then secreted into the ECM via packaging into vesicles by the glogi apparatus
What are the second steps in assembly of collagen?
Peptidase enzymes remove extension peptides, tropocollagen spontaneously forms fibrils - stabilised by covalent cross links
How does formation of cross links between tropocollagen in collagen fibrils occur?
Lysine or Hyrdoxylysine residues can be deaminated to form allysine by lysyl oxidase, covalent bonds can then spontaneously from between allysine-allysine or allysine-lysine, these covalent bonds are important in stabilising fibril structure and giving collagen its structural strength
What is the role of collagen in bone?
Collagen fibrils provides the structure onto which calcium phosphate is deposited, staggered arrangement leaves small gaps, position of the gaps (and thus a regular arrangement of fibres) is important to get smooth and regular deposition of calcium phosphate which gives bones strength
How are collagen fibrils arranged in tendons?
Fibrils are bundled into fibres which are arranged together to form the tissue
Give 2 examples of when collagen must be broken down and re formed?
1) growth and tissue remodelling (pregnancy/after birth)
2) tissue repair (scar tissue contains lots of collagen)
What is the collective name of the enzymes that break down collagen molecules?
Collagenases (metalloproteinases requiring Zn)
Why are collagenases important in cancer?
Tumours cells produce large amounts of collagenases which are involved in tumour invasion and metastasis
What is Dupuytren’s contracture and what is used to treat it?
Disabling condition where excess collagen production affects the connective tissue of the hand, treated using collagenases
What is Osteogenesis imperfecta?
a range of inherited diseases affecting bone and joint development and strength, caused by malformation of type 1 collagen. Severity of the disease varies with different forms, Type 2 is most severe causing death before or around birth and Type 1 is least severe
What is Ehlers-Danlos syndrome?
a range of inherited disorders affecting the skin and joints, major symptoms include fragile stretchy skin and hyperextandable joints, caused by a variety of defects in collagen synthesis eg. patients with Type 4 lack lysyl oxidase (Can’t form correct cross links)
How can enzymes be used in diagnosis or prognosis?
1) Used as diagnostic reagents eg. Presence in the blood may indicate tissue damage
2) Used as biomarkers of disease eg. enzyme activity may be increased/decreased/absent in some disease
3) Enzymes can be used as therapeutic agents eg. collagenesterases in dupuytrens contracture
How do enzymes alter reactions?
Increased rate of reaction but don’t shift position of equilibrium - reach equilibrium faster
How do enzymes work?
Lower the activation energy
What is meant by an energetically favourable reaction and why might an enzyme still be required for the reaction to take place?
Exothermic reaction, products at lower energy level than the substrates. May still need an enzyme as reaction could have a v. high activation energy (enzyme lowers this)
How do enzymes reduce activation energy?
1) Provide catalytically competent groups (bit that does reaction chemistry)
2) Bind substrates in such a way as to orientated them top opsonise the reaction
3) By preferentially binding and stabilising transition states of the substrate (unstable intermediate could go forwards to form product or collapse back to substrate, enyzyme stabilises it and makes it form product
What is the active site of an enzyme?
3D entity comprising crucial amino acid residues, region of binding and conversion to product, substrate binds via multiple weak interactions because its easy to unload
What amino acids residues make up a common triad in serine proteases?
His-Asp-Ser
What factors affect rates of enzyme catalysed reactions and how?
1) Substrate conc. - higher [S] = higher V
2) Temperature - optimum temp, below = low kinetic energy, above = dentures
3) pH - all enzymes have an optimum pH at different pH amino acid residues in active site may be altered
What happens in irreversible enzyme inhibition?
Covalent modification of amino acid see chains in the active site. Eg. Snake venom irreversibly blocks the active site of acetyl cholinesterase at active sites but parathion (insecticide) only blocks it reversibly
What are the 2 types of reversible inhibition?
1) Competitive
2) Non-competitive
How can competitive inhibition of an enzyme be overcome?
Increasing substrate concentration
What is Vmax?
Theoretical maximum rate of reaction
What is Km?
Substrate concentration at which the reaction rate is half its theoretical maximum value, its a measure of the affinity of an enzyme for its substrate - lower Km = higher affinity
What happens to Vmax and Km in competitive inhibition?
Vmax is unchanged and Km is increased (need higher [S] to reach V=Vmax/2)`
What is non competitive inhibition?
Inhibitor binds at independent site to substrate, alters the conformation or accessibility of the active site
Is non competitive inhibition affected by [S]?
No
What happens to Vmax and Km in non competitive inhibition?
Vmax decreased (proportion of enzyme switched off) and Km is unchanged (proportion of enzyme ‘switched on still has same affinity for substrate)
What is an IC50 value and what is it used for?
Used for comparing inhibitors, its a the concentration at which an inhibitor knocks out 50% of the enzyme, lower IC50 = more effective inhbitior
What is methotrexate and what does it inhibit?
Its a cancer drug which inhibits dihydrofolate reductase (necessary for synthesis of purines of DNA) - cancer cells are proliferative - so target this as they will be disproportionately affected
What are COX 1 and COX 2 enzymes?
Cyclooxygenases which catalyse the production of pro-inflammatory molecules prostaglandins
What is the difference between mode of action of Aspirin and Ibuprofen in terms of inhibition?
They are both NSAID’s which competitively block COX 1 but aspirin blocks it irreversibly (covalent modification of serine residues of active site) and ibuprofen blocks it reversibly
In the Lineweaver-Burk double reciprocal graphs what do the intercepts mean?
y intercept = 1/Vmax
x intercept = -1/Km
What is the Michaelis Mennen equation used to calculate?
Tells us how to work out Km ie. That [S] @ V = Vmax/2 = Km
What is Michel’s constant?
Km
How are metal ions used as co factors in enzyme function?
Can form essential components in enzyme active sites and be involved in chemistry
Eg. May bind reactants electrostically or may act as oxidising agents (electron acceptors) as they have a positive charge
What is Fe3+ an important co factor for?
Monoamine oxidase, substrate L-DOPA, involved in dopamine synthesis
Give 3 common examples of water soluble co-enzymes and there rough role?
Don’t have enzyme activity them selves but provide some sort of essential function, generally act as carriers
1) NADH and FADH2 - carry electrons (carry a ‘reducing power’
2) Co-enzyme A - carries acyl units (Carbon units from one reaction to another)
3) Biotin and Thiamine pyrophosphatase - carry CO2 units (bound to carboxylases which catalyse the incorporation of CO2 molecule into organic substrate
What has the dietary vitamin niacin got to do with co enzymes?
Forms part of NADPH, NADH - the niacin part carries the electrons and therefore the rescuing power - the bit that does the important reaction chemistry
What is the important substance in co-enzyme A?
Pantothenic acid
What is G6PDH and what does it do?
G6PDH produces a large proportion of the bodies NADPH which is needed to drive biosynthesis of nucleic acids (eg. Dividing cells and healthy growth) and lipids
What is glucose 6 phosphate? (G6P)
When glucose enters a cell, it has a phosphate added making G6P activating glucose so it can be used in glycolysis
Where does the Penrose phosphate pathway principally occur?
In the liver
Where does the Pentose phosphate pathway principally occur?
In the liver
What is the Pentose pathway?
Pathway that makes 1 of 2 things, either Ribose-5-phosphate or NADPH, substrate is glucose-6-phosphate
In which stage of the Pentose pathway is NADPH produced and what enzyme is involved?
1st stage, enzyme of G6PDH
How is the activity of enzyme G6PDH controlled?
If cells don’t have enough NADPH, NADP+ levels rise, NADP+ signals back and causes increased activity of G6PDH to produce NADPH
What are the 3 types of genetic mutation in G6PDH?
1) Destabilise the trimmer
2) Disrupt NADP+ binding
3) Disrupt activity directly
What is Glutathione molecule?
Its involved in a protective pathway that occurs in all cells, it acts as a scavenger and reacts with any reactive species eg. H2O2 and other kinds of free radicalsinhib and gets rid of them before they damage cells - they would otherwise attack the lipids of plasma membrane, destroying membrane
What is the role of NADPH in the activity of Glutathione?
Need reduced form of Glutathione to react with reactive species - to regenerate reduced Glutathione need glutathione reductase and co enzyme NADPH
What is primiquine?
Drug used to treat malaria
Why does primiquine induced haemolytic anaemia occur?
People with genetic deficiency in G6PDH don’t produce sufficient levels of NADPH so can’t regenerate enough reduced Glutathione and combat nasty substances.
In other cells this doesn’t matter as have other pathways to produce NADPH (which occur in the mitochondria) as RBC’s have no mitochondria, they can only use this pathway.
Primiquine produces lots of H2O2 which can’t be coped with by the glutathione system in RBC’s and lots of RBC’s dies
What is favism?
Deficiency in G6PDH named after the fava bean
What are ‘serpins’?
Serine protease inhibitors
What are serine proteases?
Family of proteases with serine in the active site (his-asp-ser), serine plays a crucial role in reaction chemistry
Give 3 examples of common ‘serpins?
1) a1-antitrypsin (aka anti-elastase): inhibits elastase, a protease which breaks down connective tissue, attacking things like collagen
2) pancreatic trypsin inhibitor: found in pancreas to prevent autodigestion
3) Antithrombin III: switches off blood coagulation system
In terms of control of enzymes what is an allosteric effect or?
Effector that controls how active any enzyme is by binding to the enzyme at a sight other than the active site and causing a conformational change, the sight it binds to is called the allosteric sight
In terms of control of enzymes what is meant by feedback regulation and what does it ensure?
Product of last reaction in pathway controls the activity of the enzyme in the first reaction, system is finely tuned to optimise levels of all intermediates in the pathway
In terms of control of enzyme pathways what is meant by reciprocal control?
If a pathway makes 2 different products A and B, then product B may act to stimulate the enzyme that forms A and inhibit the enzyme that forms B in order to get balanced levels of both products (eg. Nucleic acid synthesis)
Name the most common form of covalent modification used to control enzymes in metabolic pathways?
Protein phosphorylation
What is the process of protein phosphorylation and which enzymes are involved?
Gamma-phosphate of ATP is transferred to a serine or threonine residue of the enzyme we want to control
Protein kinases are involved in phosphorylation
Phosphatases are involved in dephosphorylation
Phosphorylation of an enzyme can lead to either increased or decreased activity of an enzyme
How does protein phosphorylation alter enzyme activity?
Addition of a charged group can cause a conformational change in an enzyme or modulate activity via a change to the active site
What role does protein phosphorylation have in controlling blood sugar?
Glucagon stimulates protein kinase (phosphorylase kinase) which phosphorylates ‘glycogen phosphorylase’ the enzyme involved in the last stage of glycogenolysis
Insulin hormone stimulates phosphatase enzyme which removes the phosphate group from glycogen phosphorylase inactivating it
What is proteolytic activation?
Full formed and folded protein, that is activated when it is cut by a proteolytic enzyme, either by cutting the polypeptide chain or cutting out a section of the chain which causes the protein to become active!
What is a zymogen?
Pre cursor protein, that is fully formed but inactive, also called a pro enzyme (needs to be proteolytically cleaved)
After proteolytic activation can an enzyme be inactivated?
Proteolytic activation is irreversible, to stop enzyme activity the enzyme would have to be destroyed
Name 2 common enzyme controlled pathways which are controlled by proteolytic activation?
1) Digestive enzymes (pancreatic proteases)
2) Blood clotting cascade
Where does the specificity of serine proteases come from?
Specificity comes from the precise shape and chemical character of substrate binding pocket not from the active site
Name 3 serine proteases and how they differ?
1) Chymotripsin: cleaves c terminal of hydrophobic residues
2) Trypsin: cleaves C terminal to Lys and Arg
3) Elastase: cleaves C terminal to Gly and Ala
How is digestive enzyme chymotrypsin proteolytically activated?
Trypsin cleaves to partially active chymotrypsin which then auto cleaves to fully active chymotrypsin, fully active (mature trypsin) is made up of three sections of polypeptide chain joined by disulphide bonds
How is digestive enzyme trypsin proteolytically activated?
Remove of amino acids 1-6 causes activation, initial activation by duodenal enzyme enteropeptidase and then self activating
Name 3 enzymes which are proteolytically activated by trypsin?
1) Chymotrypsin
2) Elastase
3) Carboxypeptidase
What is the role of Vitamin K in the blood clotting cascade?
Prothrombin is carried on platelets to the site of injury where it is activated by factor 10a, prothrombin sticks to the platelets via calcium (acts as glue) and is required to have gamma-carboxyglutamates (amino acids that are good at binding calcium) to bind the calcium and stick to the platelet. gamma-carboxyglutamates are formed in a Vitamin K dependent reaction, can have bleeding disorders in vitamin K. Structural analogues of Vitamin K can be used as anti coagulants
How does the lysis of blood clots occur?
1) Plasminogen is cleaved to plasmid by TPA (Tissue type plasminogen activator)
2) Plasmin is then an active serine protease which cleaves fibrin clot to peptides
TPA adheres to the clot and binds plasminogen
TPA can be used as a therapeutic agent (can be recombinant TPA or streptokinase (bacterial enzyme with similar activity)
In terms of enzymes as diagnostic markers what are izoforms?
Enzymes have different izoforms which originate from different tissues - need to be able to identify the izoform to be able to identify the tissue that it came from
In what condition is serum amylase raised?
Acute pancreatitis
In what condition is serum acid phosphatase raised?
Prostatic carcinoma
In what condition is serum alkaline phosphatase raised?
Liver disease, osteoblastic bone disease
In what condition is serum GOT (glutamate oxaloacetate transaminase)/AST raised?
MI and liver damage
In what condition is serum GPT (glutamate pyruvate transaminase raised)/ALT raised?
Liver cell damage
In what condition is serum LDH (lactate dehydrogenase) raised?
MI
In what disease is serum creating kinase raised?
MI, Skeletal muscle disease
Why does detection of abnormal enzymes in the serum normally indicate tissue damage?
Cytosolic enzymes released - slight damage
Mitochondrial enzymes released - severe damage
Why can some enzymes be detected in the blood stream after damage than others?
Because all enzymes have different half lives in the blood, some are raised immediately after damage, some take longer
What is the standard unit of measurement of enzyme activity?
International Unit (IU), 1 IU = amount of activity that will convert 1 microcode of substrate per minute, normally given as a function of the tissue its being measure in eg. 1 IU/ml serum
Which 3 substances are detected in blood serum after to liver damage and in what order?
Raised levels of GPT (more highly raised) and GOT will be seen followed by a rise in bilirubin
What does GPT/ALT do in the liver?
Glutamate + PYRUVATE -> Alanine + a-ketoglutarate
What does GOT/AST do in the liver?
Glutamate + oxaloacetate -> Aspartate + a-ketoglutarate
Instead of serum levels of acid phosphatase what substance is more commonly used to indicate prostatic carcinoma?
Prostate specific antigen (PSA)
Which 3 substances are detected in the blood post MI and in what order?
1) Peak of creating kinase
2) Followed by a peak of GOT/AST
3) Followed by a peak of lactate dehydrogenase
What is the role of creatine kinase in the body?
Creatine + ATP creatine phosphatase + ADP + Pi
Reaction is catalysed by creatine kinase when ATP is plentiful, phosphocreatine then acts as an emergency energy supply for muscle
Which 4 substances are detected in the blood post MI and in what order?
1) Peak of creating kinase
2) Followed by a peak of GOT/AST
3) Followed by a peak of lactate dehydrogenase
4) Cardiac specific protein Troponin 1 (cTn1) also supports diagnosis of MI
What are the 2 different izoforms of creatine kinase and where are they found?
CK-MB (more specific to cardiac tissue - has very low presence in blood normally)
CK-MM (more specific to muscle tissue - predominant izoenzyme in the blood)
Need to do electrophoresis to find % of different izoenzymes
What is the normal range of CK?
60-400 microliters
What is the difference between different izoenzymes of the same enzyme?
1) Have same substrate specificity
2) Different kinetic properties (Km)
3) Can be regulated differently - different sensitivity to different inhibitors
4) Often show different preferences for driving a reaction one way or another
What are the differences in structure between the different izoforms of the same enzyme?
Related forms of a protein but coded for by different genes, have very similar polypeptide chains with only a few key differences accounting for different properties
What are the 5 different izofroms of LDH and where are they found and when are they elevated?
1) H4 (LDH1) found in the heart (post MI)
2) H3M (LDH2) found in monocytes/macrophages (serum normally only contains this form
3) H2M2 (LDH3) found in lungs (pulmonary disease)
4) HM3 (LDH4) found in kidney, pancreas
What are the 5 different izofroms of LDH and where are they found and when are they elevated?
1) H4 (LDH1) found in the heart (post MI)
2) H3M (LDH2) found in monocytes/macrophages (serum normally only contains this form
3) H2M2 (LDH3) found in lungs (pulmonary disease)
4) HM3 (LDH4) found in kidney, pancreas
5) M4 (LDH5) found in liver, skeletal muscle
What does LDH normally do?
Catalyse the reaction:
PYRUVATE lactate
Depending on the izoform has a preference for one way or the other
What is the difference between the H and M forms of LDH?
H form is better at converting lactate to PYRUVATE - evolved to allow the heart to do this during severe exercise
M form is better at converting PYRUVATE to lactate - muscles do this during anaerobic exercise
What is the difference between the H and M forms of LDH?
H form is better at converting lactate to PYRUVATE - evolved to allow the heart to do this during severe exercise
M form is better at converting PYRUVATE to lactate - muscles do this during anaerobic exercise
How do different forms of LDH help the muscles and heart function?
When lactate is converted to PYRUVATE is converted to lactate in anaerobically repairing muscles NADH is produced, as no O2 then the ETC is at a standstill so NADH can’t be used in the anaerobically repairing muscles, as O2 is never limiting in the heart etc. the NADH is shuttled to them and the reaction lactate to PYRUVATE to release the NAD+ is favoured
NADH basically shuttled from where it can’t be used in the muscles to where it can be used in the heart
In terms of LDH what is the M:H ratio?
ratio of M4 type activity to other forms
In terms of LDH what is the LDH1:LDH2 ratio used for?
This is normally low but flips to >1 post MI
What tests are becoming more common post MI and what is there advantage?
Immuno assays for CK, myoglobin and cardiac troponin 1 (heart muscle filament protein), dont measure enzyme activity but measure amount of enzyme, there cheap and reliable (use recombinant DNA)
How are enzymes used in the treatment of childhood acute lymphoblastic leukaemia?
In normal cells asparagine is non essential as it can be synthesised by the enzyme asparagine synthetase, tumour cells are deficient in this enzyme so can only get asparagine from the diet - if you reduce supply then they are prevented from synthesising proteins and thus growing. Deliver asparaginase (which breaks asparagine down into aspartate and NH3) to reduce supl of asparagine to tumour cells. The name of the drugs are ELSPAR or KIDROLASE
What are anti neoplastics?
Name used to refer to drugs which inhibit or prevent the growth and spread of tumours or malignant cells
How are enzymes used as thrombolytic agents?
TPA (tissue type plasmin activator) which proteolytically activates plasmin is produced using recombinant DNA (Alteplase, rTPA, Actilyse) and is administered following coronary thrombosis
How are enzymes used as detoxifying agents in kidney dialysis?
Urea builds up in people with non functioning kidneys, urease enzyme in the dialysis membrane breaks the urea (+H2O) down into ammonia and CO2, the ammonia is the removed by being absorbed by activated carbon
What is enzyme replacement therapy?
When patients are administered enzymes which they are deficient in due to a genetic defect
What is an enzymopathy?
loss of enzyme activity due to a genetic defect
What is acatalasaemia?
enzymopathy where you’re deficient in catalase
What is Haemophilia A?
Enzymopathy where you’re deficient in Factor 8
What is Lesh-Nyhan syndrome?
Enzymopathy where you’re deficient in HGPRT (leads to build up of uric acid in body fluids)
What is Phenylketonuria?
Enzymopathy where you’re deficient in phenylalanine hydroxylase
What is the treatment for paracetemol poisoning and why must paracetemol levels in the blood be measured before its administered?
Treatment is n-acetyl/cysteine/methionine, its a compound which can have a toxic effect so a precise dose is required according to the level of paracetemol in the blood
How are enzymes used to measure paracetemol levels in the blood in paracetemol poisoning?
bacterial enzyme is administered which converts paracetemol to acetate + p-aminophenol, another reagent is then added which converts the p-aminophenol into a coloured dye product
At low levels how is paracetemol metabolised?
Paracetemol is conjugated with glucuronide or sulphate and renal excreted in the urine (no involvement of cytochrome p450)
At high levels of paracetomol in the blood how is paracetemol metabolised and what is the issue with this, why can it lead to toxicity?
At high levels the normal system is overloaded so paracetemol instead gets broken down by cytochrome p450 to a quinone derivative, some of that is excreted via the glutathione system, but there is not enough glutathione to get rid of it all so the bit left over attacks liver cells compromising liver function
How does the treatment for paracetemol poisoning (N-acetylcysteine) work?
N acetyl cysteine boosts the amount of glutathione available in the body so all of the toxic quinone derivative can be removed
How are enzymes used to measure metabolites in the blood?
1) Metabollite specific enzyme which generates H2O2 as a bi product
2) Add a second peroxidase enzyme which reacts H2O2 molecule with a dye (chromagen) to form a coloured product (spectrophotometer measures colour change)
How are enzymes used in the measurement of blood glucose?
1) Glucose oxidase makes H2O2
2) Peroxidase reacts H2O2 with a dye (chromagen) to make a coloured product
What are immobilised enzymes and what are they used for?
Plastic strip containing immobilised enzymes and colour reagents (chromagen), rapid, semi quantitive test for different compounds eg. urea, total protein, glucose, pH, cholesterol, alcohol
What is Lesh-Nyhan syndrome?
Enzymopathy where you’re deficient in HGPRT (leads to build up of uric acid in body fluids). inherited disorder in nucleotide biosynthesis, affected children have severe neurological and behavioural problems
What are enzyme electrodes and how do they work?
Have an enzyme in an electrode trapped by a permeable membrane, analyte comes into contact with the enzyme and makes an ionic product, electrical conductivity of the solution changes in proportion to the concentration of ionic product - measurable signal that reflects the concentration of the metabolite. Fast, sensitive, used in labs to measure stuff eg. glucose, cholesterol etc.
Why is enzyme replacement therapy difficult in Lesh Nyhan syndrome?
Because the enzyme that is absent (HGPRT) is normally present within the cell and you can’t get enzymes into the cell, gene therapy is a better avenue
What disease does deficiency in Riboflavin (B2) lead to?
1) Skin lesions on ears
2) Angular stomatitis
3) Glossitis
4) Cheilosis
5) Dermatitis
What disease does deficiency in Niacin cause?
Pellagra (diarrhoea, dermatitis, dementia)
What disease does deficiency in Thiamine (B1) cause?
Beri Beri (weight loss, heart problems, neurological dysfunction)
What disease does deficiency in Ascorbic acid (Vitamin C) cause?
Scurvy (general weakness, fatigue, easily bruised skin, spontaneous bleeding, failure of wounds to heal, bleeding gums)
What disease does deficiency in Biotin cause?
Rash about eyebrows, Muscle Pain, Fatigue
What disease does deficiency in globalamin (B12) cause?
Pernicious anaemia (too big RBC’s with too little Hb), Methylmalonic acidosis (build up of methylmalonic acid in the blood)
What disease does deficiency in folic acid cause?
Anemia, neural tube defects in development
What disease does deficiency in folic acid cause?
Anemia, neural tube defects in development
What disease does deficiency in Pyroxidal (B6) cause?
Depression, confusion, convulsions
What disease does deficiency in pantothenic acid cause?
Hypertension
What is wrong with the collagen formed in patients with scurvy?
Low in Hyp (forms H bonds) and Hyl (forms covalent cross links between chains and glycosylation sights)
Collagen formed is low in cross links and loses its strength, bones and other stuff can rupture and break
Why do patients with scurvy also suffer with fatigue?
Enzymes that synthesise carnitine (transfers fatty acids into mitochondria to provide a source of energy) also requires ascorbic acid as a co factor
What are the 3 main types of protein?
1) globular proteins: enzymes, Hb, compact soluble
2) fibrous proteins: keratin, collagen, elastin, elongated, insoluble due to high hydrophobic amino acid content
3) membrane proteins eg. ion channels
What is haemolytic anaemia?
Condition in which red blood cells are destroyed and removed from the blood stream before their normal lifespan is over
What are the advantages of keeping haem unit within a protein molecule?
1) Fe atoms are kept in +2 form
2) Binding of small molecules eg. CO is inhibited
What are myoepithelial cells?
epithelial cell with contractile function
