Enzymes Wk 3 Flashcards
What do enzyme do
Catalyse biochemical reactions but changing substrates into products but without changing themselves
What does the tertiary structure in enzymes dictate
The active site where the substrate binds
What binds in allosteric sites on enzymes
Regulatory molecules
Isoenzymes
Causes by coding gene variants and have different physical properties
Isoforms
Due to post translational modifications but same genetic coding
When are enzymes released in the plasma
In cellular degradation
After tissue injury
Are enzymes specific to plasma or tissues
Enzymes can be specific to plasma like coagulation factors or present in one or more tissues
How are enzymes measured
By the production or consumption of a measurable substance so it is not direct
What is the point of a indicator reaction
To use the product from the assay reaction to form another produce which is easy to measure
What is in the assay reaction
The enzyme we are interested in
The product of this reaction is used as a substrate in the indicator reaction
Why are enzymes helpful energy wise
Substrates need a lot of energy to become a product
When combined with an enzyme the amount of total energy needed is less to get to the same product so the rate of reaction is increased
What does Vmax mean
Maximum velocity of the reaction
All of the enzyme is saturated with substrate, adding more substrate will not do anything
What does Vmax depend on
The affinity between the substrate and the enzyme
What is the Km
The substrate conc where the reaction velocity if half the maximum
A measure of substrate binding affinity
What does first order kinetics depend on
The rate depend on the substrate conc
Increase substrate = increase speed as the reagent is limiting
What does the rate of zero order kinetics depend on
The rate is independent of the substrate conc
Reaction is as fast as it can be, increase the substrate concentration will do nothing for speed
3 patterns for enzyme reactions
- Linear, proportional increase as rate is constant
- Decreases over time
- Lad phase, linear, curves off
When should you measure absorbance in a linear graph
End point assay
Velocity = max rate (k) so can use any two points
When should you measure abs in a decreasing graph
Velocity never reaches a maximum
Need to optimise reaction by changing temp, changing buffer, adding more substation conc, secondary reactions to get rid of inhibitory products
When to measure absorbance in sigmoidal curve with a lag phase
Max rate is in the middle
Continuous assay - measure linear part
Most classic curve
What happens in the lag phase
First reaction in the coupled ones are happening so forming the needed substrate
Waiting for optimal temp and pH
What are cofactors
Non protein molecules or irons that activate or enhance enzyme function
Cofactor examples
Mg2+
NAD+
ATP
What is a coenzyme
Organic non protein molecules that bind with the protein to form an active enzyme
Co enzyme example
Vitamins
Apoenzyme and holoenzyme define
Proteins are apoenzymes
Holoenzymes are activated enzymes made when the coenzyme bind with the apoenzyme
What are activators
Small regulatory molecules or ions that increase reaction rate
What do activators promote
Formation if the most active state of the enzyme/substrate by binding to a different site (allosteric)
What can inhibit activators
Additives like Mg/cofactors
What are inhibitors
Small regulatory molecules or ions that decrease reaction rate
Competitive inhibitors
Competes with the substrate for active site binding
Non competitive inhibitors
Allosteric
Binds to a different site on the substrate, not the active site and prevents the enzyme from releasing products
Units for enzyme measuring
IU or U
U/L
As we focus on activity not mass
What reaction does creatine kinase catalyse
The reversible phosphorylation of creatine by ATP
What inhibits the creatine kinase catalyst reaction
Metal ions but needs Mg
Where is creatine kinase found, order of most abundant
Skeletal muscle
Heart
Brain
GI
Urinary bladder
When is creatine kinase elevated
In muscle damage
Myocarditis, muscular dystrophy, crush injuries
To measure creatine kinase we do the reverse reaction from phosphocreatine to creatine, why
It is 6x faster than the forward reaction
Assay for creatine kinase measures what
NADPH at 340nm
What molecules are in excess in CK
Everything but CK so this is the rate limiting factor
Except the substrate we produce and then those products
What is the most heart specific CK iso enzyme
CKMB
rises 4 hrs after MI
Gone within 3 days
Sandwich immunoassay to find it
Is CK seen in myocardial infarction
Yea
When is CKBB seen
Brain
Bladder
Bowel - UT
When is CKMM seen
Muscle injuries
Sandwich assay order
Magnet binds with capture antibody
Antigen binds
Detector antibody
Secondary antibody with conjugate
Substrate with colour change
What are aminotransferases
Transfer amino groups from amino acids to make alpha keto acids and back again
Where is aspartate aminotransferase (AST) found
Heart
Liver
Skeletal muscle
Kidney
When does aspartate AT increase
Liver disorders
Muscle damage
PE
Muscular dystrophy
Haemolysis
What does the aspartate AT assay measure
Measures the consumption of NADH decreasing as NAD+ is colourless and hard to measure
Assay reaction for aspartate
Making oxaloacetate
Indicator reaction for aspartate
Dehydrogenase reaction
Using MD
Where is alanine aminotransferase
Liver and kidney
Cytoplasmic - aspartate has cytoplasm and mitochondrial forms
When does alanine (ALT) increase
Liver disorders
How much depends on tissue damage
High ALT- need to check aspartate too
What is measured in the ALT assay
Rate of NADH decrease is measured photometrically at 340nm
What is alkaline phosphotase (ALP)
A hydrolyse that dephosphorylates molecules at alkaline pH
What activates alkaline phosphatase
Divalent carions
Zn
Mg
What inhibits alkaline phosphatase
Anions
Grey top
Phosphate
Cyanide
Where is alkaline phosphatase found
Everywhere
Kidney
Bone
Liver
Placenta
When does alkaline phosphatase increase
Pregnancy, healing, growth
Bone disease
Liver disease (hepatitis, cirrhosis)
what do GGT do
Transfers a gamma glutamyl functional group from peptides to an acceptor
When goes GGT/gammaGT increase
Hepatobiliary disease
Drug metabolism
Cancer
Alcohol
Obesity
Pancreatitis
Where is gammaGT found
Cytoplasmic
Kidney
Liver
Pancreas
Intestine
Why is gammaGT critical for
Maintaining levels of reduced glutathione
What decreases gammaGT function
Citrate
Oxalate
Fluoride
By 10-15%
What does LDH catalyse
The oxidation of lactate to pyruvate
Where is LDH found
Cytoplasmic
Highest:
Liver
Heart
Kidney
Muscle
RBC
What inhibits LDH
EDTA
When does LDH increase
Myocardial infarction
Liver disorders
Haemolysis
Tumour lysis syndrome
Cancers some
Why can LDH be good to measure
In tumour can see if tumour is working
More tumour cells dying = LDH increasing
Why is LDH measured less
More specific tissue markers are available
What does amylase catalase
The degradation of polymeric carb
When does amylase increase
Pancreatitis
Biliary tract disease
Intestinal obstruction
Peptic ulcers
When does salivary amylase increase
Salivary glands inflammation
When does pancreatic amylase increase
Acute pancreatitis but lipase is the preferred test as amylase slips into urine and cannot be monitored as well
Alpha and gamma amylase found in
Animals
Can amylase be in pee
Yes
Alpha and beta amylase found in
Planta
What does lipase hydrolyse
Long chain fatty acids into short chain fatty acids and acyl glycerol
Is lipase found in urine
No it is filtered and reabsorbed
Where is lipase found
Pancreatic acinar cells
GI Mucosa
How is lipase used for acute pancreatitis diagnosis
If the levels of lipase are 3x the upper limit of normal it is likely
When does lipase peak
24h
Decreased within 7-14 days
More specific and stable than amylase
