Enzymes- Introduction to enzymes, regulation of enzyme activity and enzymes in diagnosis, prognosis and therapy Flashcards
What are enzymes and what do they do?
- Biological catalysts of chemical reactions
- Specific action on particular biochemical compounds (substrates)
- Nearly all are proteins
- Increase the rate at which the reaction equilibrium is reached, but do NOT shift the position of equilibrium
Activation energy
Difference of free energy
Transition state
The highest energy
How do enzymes decrease EA?
- by providing catalytically competent groups for a specific reaction mechanism
- by binding substrates such that their orientation is optimised for the reaction
- by preferentially binding and stabilising the transition state(s) of the reaction
What is the AS of an enzyme
- is the region of the enzyme at which substrate binding and conversion to product takes place;
- is a 3-dimensional space comprising crucial amino acid residues;
- may represent only a small part of the protein structure;
- binds substrate via multiple weak interactions;
- provides substrate specificity because of its unique 3D arrangement of atoms
How is enzyme activity measured?
Use the Michaelis- Menten model
Km
Substrate concentration at which the rate is half Vmax
Vmax
Maximum rate of reaction
Factors affecting enzyme-catalysed reaction rates
- Substrate/enzyme concentration
- Temperature
- pH
- Inhibitors, both natural and exogenous
Irreversible enzyme inhibition
Covalent modification of the enzyme, usually at amino acid side chains in the AS
Competitive inhibition and how Km is affected
enzyme
• Inhibitor usually binds same site as substrate (active site)
• Inhibition can be overcome by high substrate concentration
• In the plot for double reciprocal, the lines have the same Vmax because the inhibitor gets swamped by the substrate concentration. However, Km is increased – more substrate is needed to reach half of Vmax.
Non-competitive inhibition and how Km is affected
- Inhibitor and substrate can bind simultaneously at independent sites
- Inhibitor alters conformation or accessibility of active site
- Inhibition not affected by high substrate concentration
- Vmax decreases (some of the enzyme unusable), Km is unchanged , increasing substrate concentrate has no effect.
Non-steroid anti-inflammatory drug examples and what they do
Aspirin:
• Covalent modification of a serine residue in the active site
• Inhibitor binding is competitive, inhibition is irreversible
Ibuprofen:
• Binds to active site, but not covalently attached
• Inhibitor binding is competitive, inhibition is reversible
IC50
The inhibitor concentration at which 50% of activity remains for a standard amount of enzyme and substrate
Cofactors are essential for enzyme function- metal ions
- Includes major minerals such as Mg2+ and Ca2+ and trace elements such as Cu2+, Zn2+, Fe2+/3+, Mn2+ and Mo4+
- Metal ions can be part of active site and/or be involved in electrostatic substrate binding (Zn2+ in carbonic anhydrase, Mg2+ in kinases)
- Metal ions may act as redox agents (Fe2+/3+ in cytochrome p450s)
- Metal ions may regulate activity of enzymes (Ca2+ in calpain)
Cofactors are essential for enzyme function- coenzymes
• Coenzymes function as carriers of reaction components:
- NADH and FADH2 carry electrons (‘reducing power’)
- Coenzyme A carries acyl units
- Biotin and thiamine pyrophosphate carry CO2 units (bound to carboxylases)
Effect of mutations in enzymes
Glucose-6-phosphate dehydrogenase (G6DPH) deficiency causes metabolic defects
- X-linked recessive; most carriers asymptomatic (disease without showing symptoms)
- The enzyme produces a large proportion of the body’s NADPH needed to drive biosynthesis of nucleic acids, lipids etc.
- Where symptomatic, symptoms can include haemolytic crises (accelerated rate of blood destruction), jaundice (can lead to brain damage (kernicterus) in infants)
Favism
A disease that results from a deficiency of the enzyme glucose-6-phosphate dehydrogenase (G6PD). Favism usually is due to a genetic disorder.
Controls of enzyme activity
- Inhibition (reversible or irreversible)
- Feedback regulation
- Covalent modification- post translation modification
- Proteolytic (breaking down proteins) activation
How does inhibition affect enzyme activity?
Serine protease inhibitors- Prevents protease from attacking tissue, controls activity of digestive enzyme
and switches off blood clotting system.
How does feedback regulation affect enzyme activity?
The enzyme may be regulated directly by the product it produces or a downstream product of its metabolic pathway.
Can amplify/ stop enzymes.
How does covalent modification affect enzyme activity?
- Protein phosphorylation is a universal mechanism of enzyme modulation
- Gamma-phosphate of ATP is transferred to amino acid residues (e.g., Ser, Thr, Tyr)
- Enzymes are phosphorylated by other enzymes, called protein kinases
- Phosphate groups are removed by phosphatases
- Charged group induces conformational change in the enzyme
How does proteolytic activation affect enzyme activity?
- Inactive ‘zymogen’ precursors (an inactive substance which is converted into an enzyme when activated by another enzyme) or ‘pro-enzyme’ are activated by proteolysis.
- Irreversible activation of enzyme after removal of part of peptide chain
Example of a serine proteases of the digestive system
Chymotrypsin
- Digestive enzyme with a zymogen precursor
- Mature active protein has 3 polypeptide chains linked by disulfide bonds
- Zymogen precursor is a single polypeptide
- Three enzymes evolved from common ancestor
- Functionally distinct enzymes
Activation of zymogens
Trypsin:
• Made as zymogen called trypsinogen
• Removal of amino acids 1-6 cause activation - causes small conformational change
Lysis of blood clots by plasmin
- Activator adheres to clot and binds plasminogen - targets activity to correct place
- Enzyme of activator cleaves plasminogen to active plasmin
- Plasmin digests fibrin clot to small peptides
- Administration of recombinant activator or can significantly increase chances of surviving coronary thrombosis- ‘Clot-busters’ or thrombolytics
Enzyme isoforms/ isoenzymes
Enzymes with similar function, and sometimes similar amino acid sequence, but expressed from different genes.
Different forms of enzyme that have the same substrate specificity but are coded for by different genes
Isoenzymes – key examples
Lactate dehydrogenase, cyclo-oxygenase, creatine kinase
What is the standard measurement of enzyme activity?
1 IU is the amount of activity that will convert 1 micromole (µmole, or 10-6 moles) of substrate per minute under standard defined conditions
Two factors for enzymes as diagnostic/ prognostic markers
- Tissue-specific enzymes (for example indicating tissue damage when found in serum
- Differential tissue distribution of enzyme isoforms
Liver function test for tissue specific distribution of isoenzymes
- Albumin- not an enzyme it is a transport protein (<3.5-5.3 g/dL) – Indicates general loss of secretory function, but decrease could also be due to kidney disease
- GOT (AST) (6-48 IU/L) – Elevated in acute liver damage, but could originate from red blood cells or muscle, so not liver-specific
- GPT (ALT) (7-55 IU/L) – A more specific indicator of liver damage (in RBC but not muscles thus can indicate damage if present in liver) than GOT, high activities tend to rule out muscle damage
- Total transaminase (GOT+GPT) – If both activities are high but with normal ALP = liver necrosis. Elevated levels could indicate alcohol toxicity, viral infection, cancer, others. Values >1000 IU/L could be paracetamol poisoning and/or severe/sudden liver failure. GOT/GPT ratio can help discriminate between liver and muscle damage
- Alkaline phosphatase (ALP) (45-120 IU/L) – If elevated, could indicate large bile duct obstruction. Caution: also high in Paget’s Disease of Bone, growing kids (bone metabolism could be high) and 3rd trimester pregnancy
- Direct (>0.4 mg/dL) and total (>1.2 mg/dL) bilirubin – Direct bilirubin is combined for excretion in the bile- if normal, jaundice results from problem upstream of the liver (some form of haemorrhage causing high total bilirubin). Elevated direct bilirubin indicates normal conjugation function in the liver, but failure to excrete (bile duct obstruction).
- Prothrombin time (INR) – slower clotting time indicates failure of the liver to synthesise and secrete blood clotting proteins such as prothrombin. Also used to monitor warfarin usage and Vitamin K status (see also topic ‘Blood’)
- Gamma glutaryl transpeptidase (GGT; 9-48 IU/L) – Sensitive to minor changes in liver functions, more ‘liver specific’ than transaminases. Raised by chronic alcohol/drug abuse.
Markers of cardiac injury
- Creatine kinase
- Myoglobin (Myo)
- Cardiac troponin-I (cTnI)
- Lactate dehydrogenase (can be used relatively late after acute myocardial infarction; AMI)
Creatine kinase isoforms- what do they do?
CK phosphorylates creatine using ATP.
Creatine-phosphate broken down with ADP to ATP for energy source and creatine.
Why is serum CK high when there is muscle damage?
Serum CK activity measurements is used in cases of crush injuries to determine severity of muscle tissue breakdown (rhabdomyolysis).
Rhabdomyolysis- muscle tissue breakdown with release of intracellular contents (myoglobin) into circulation. Elevated CK levels. Dark, reddish-brown urine due to myoglobinuria. Myoglobin may block off the structures of the kidney and break down into toxic compounds leading to acute tubular necrosis or acute renal failure.
What are Lactate dehydrogenase (LDH) isoforms and what do they show?
LDH is a tetramer of subunits.
Found in the heart.
Ratio between LDH1 and LDH2 in the blood. Usually not much LDH1, LDH2 in the blood normally.
LDH1 increases after AMI (acute myocardial infarction).
How are enzymes used as a diagnostic reagents?
- Measurement of drug levels
* Measurement of blood glucose
When does paracetamol levels in plasma become toxic?
What is the treatment before toxic and if it does become toxic?
- Treatment within 12 h allows full recovery
- After 12 h, irreversible liver damage may occur (transplant may be needed)
- Treatment with N-acetylcysteine; precise dose required- need to know how much paracetamol is circulating the body
Before-Take blood and use bacterial enzyme that converts paracetamol to other compounds.
Toxic- cytochrome P450 is used. Paracetamol is oxidised using this, to quinone derivative.
N-acetylcysteine- similar to glutathione so can be used for excretion.
Insufficient glutathione modified liver enzymes produced which leads to toxicity and death.
Names of blood glucose levels when too low and high
<0.5 mg/ml - hypoglycaemia (dangerous)
>1.8 mg/ml -hyperglycaemia (long-term damage)
How to see how much glucose is in the urine?
Coupled reaction- two different enzymes used to make a dye- intensity of blue colour shows how much glucose there is in the urine.
NADH has a fluorescence.
Enzymes as therapeutic agents in what?
(1) In cancer therapy- Childhood Acute Lymphoblastic Leukaemia (CALL)
(2) As thrombolytic agents - recombinant TPA
(3) As detoxifying agents - kidney dialysis
(1) In cancer therapy- Childhood Acute Lymphoblastic Leukaemia (CALL) how is it used?
(1) In normal cells: asparagine (Asn) is non-essential (made by asparagine synthetase)- cells can make it themselves if not from diet. Tumour cells are deficient in Asn synthetase, therefore Asn is essential.
Treatment: deliver asparaginase (breaks down Asn)
Effect: Plasma levels of asparagine are lowered, specifically affecting growth of tumour cells. It starves the tumour cells of Asn.
Enzymes as therapeutic agents- as thrombolytic agents - recombinant TPA
Lysis of blood clots by plasmin
- TPA adheres to clot and binds plasminogen - targets activity to correct place
- Serine protease activity of TPA cleaves plasminogen to active plasmin
- Plasmin digests fibrin clot to small peptides
- Administration of recombinant TPA or can significantly increase chances of surviving coronary thrombosis- ‘Clot-busters’ or thrombolytics
Enzymes as therapeutic agents- as detoxifying agents - kidney dialysis
Small reactor containing urease and activated carbon. Urease breaks down urea and ammonia is scrubbed out.
13) What type of inhibition is caused by covalent modification of amino acid side chains in the active site?
Irreversible, competitive
How is a zymogen activated
By proteolysis
