TOPIC 4 - enzymes Flashcards
do enzymes change the position of equilibrium ?
NO
increase the rate at which equilibrium reached NOT the position
what are the 3 ways/ which enzymes reduce Ea
- providing catalytically component groups for a specific reaction
- bing substrates in an orientation optimised for the reaction
- preferentially binding and stabilising the transition state of the reaction
what is the transition state?
state of forward and backward reaction
what does stabilsing the transition state mean?
highest energy barrier that needs to be overcome to make/break bonds is lowered (i.e. stabilising the transition state of the reaction), thereby making the reaction faster
what is the active site of an enzyme ?
3D space w/ crucial AA residues that are there to fold substrate/do reaction
what is the most important complex that is made in the reaction
Enzyme-substrate complex
what is trypsin?
proteolytic digestive enzyme
how many AA are in the active site of trypsin and how are they arranged
3
the residues come togther to make the active site ‘niche’ for binding substrate
what is the equation for Michaelis constant ?
Km= (K2+K3)/K1
what is the assumption made for the Michaelis constant
K3 «_space;K2
so K3 becomes unimportant
what is the Michaelis-Menten equation and what do the symbols stand for?
V= Vmax* [S]/([S]+Km)
V- rate of reaction
Vmax- max theoretical rate of reaction
what happens if [S]»Km
highest rate of reaction
value close to 1
what happens if [S]
slow/no reaction
value close to 0
what happens if [S]=Km
rate of reaction becomes half V max
what factors affect enzyme-catalysed reactions ?
- substrate/enzyme conc
- temp
- pH
- inhibitors
what are the 4 types of inhibitors?
- reversible (usually non-covalent)
- irreversible (usually covalent)
- competitive
- non-competitive (or uncompetitive)
does the Vmax and Km value stay the same in completive inhibition?
Vmax stays the same
Km not the same
does the Vmax and Km value stay the same in non completive inhibition?
Vmax not the same
Km the same (as its a measure of the affinity of enzyme for the substrate )
what does methotrexate inhibit?
dihydrofolate
what is methotrexate used for at high conc and why
cancer drug
reduces dihydrofolate = reduces amount of DNA replicated
what kind of inhibition does aspirin have
competitive and irreversible (covalent modification)
what kind of inhibition does ibuprofen have
competitive and reversible (non-covalent binding )
what are the 2 classes of co factors essential for enzyme function
metal ion
coenzymes
what metal ions are cofactors?
Mg2+ Ca2+ Cu2+ Zn2+ Fe (2+/3+) Mn 2+ Mo 4+
how can metal ions be involved in enzyme action
- part of active sites
- involved in catalysis
involved in electrostatic substrate binding - act as REDOX agents (Fe2+/3+)
- regulating activity of enzyme (Ca2+ in calpain)
what electrostatic substrate binding is Zn2+ involved in?
- Carbonic anhydrase needs Zn2+ as cofactor
- Zn2+ in active site and binds to water
explain how Zn2+ is used as a cofactor in carbonic anhydrase
- Zn2+ in carbonic anhydrase active site binds to H2O and activates it for reaction with CO2
- CO2 transported as bicarbonate as it reacts with water (can’t really bind on its own)
- Carbonic anhydrase uses zinc to convert CO2 and H2O to carboxylic acid
- Carboxylic acid then dissociates a H+ which binds to Hb
- Hb is protonated = causes release of oxygen in tissues
what are essential components of coenzymes?
water soluble vitamins
what vitamin and coenzyme function is stomatitis due to?
- Riboflavin (B2)
- FAD,FMN
what vitamin and coenzyme function is pellagra due to?
Niacin
- NAD+, NADP+
what vitamin and coenzyme function is WKS/high output heart failure due to?
Thiamine
Thiamine pyrophosphate
what does Biotin deficiency cause and problems with what co enzyme?
- biotinylated carboxylates
- alopecia, dermatitis, herding and vision problems
what 2 vitamin deficiency cause anaemia?
- cobalamin (B12) (cob amide coenzymes)
- folic acid (tetrahydrofolate)
what vitamin and coenzyme function is peripheral neuropathy due to?
- pyridoxal (B6)
- pyridoxal phosphate
what is tryptophan?
precursor for niacin
what are the molecules involved in the steps in the production of NAD+?
1- tryptophan
2- Niacin (B3/nicotinic acid)
3- nicotinamide (intermidiate for NAD+ production)
4- NAD+
what is the presentation of pellagra
3 D’S
dementia, diarrhoea, dermatitis
what’s another condition where we see pellagra
chronic alcoholicism
what does pellagra cause?
malnutrition / malabsorption
highlight the steps in which alcohol/ethanol is broken down to acetate
1- ethanol broken down by alcohol dehydrogenase
2- reduction reaction : NAD+ –> NADH
3- = acetaldehyde
4- this is broken down by acetaldehyde dehydrogenase
5- reduction reaction
6- = acetate
what organ metabolises ethanol?
liver
why does acetaldehyde need to be broken down?
reacts with our own proteins and causes headaches
what is the most common enzyme deficiency ?
G6PDH deficiency
is G6PDH deficiency a sex linked disease?
yes
X-linked (most carries asymptomatic0
what is G6PDH used for?
production of NADPH needed for biosynthesis
what are the symptoms of G6PDH
most carries asymptomatic
- where symptomatic:
- haemolytic crisis
- jaundice
what 2 pathways can oxidised glucose (Glucose-6-phosphate) go down?
- glycolysis
- pentose phosphate pathway
what 2 things do the Pentose phosphate pathway produce?
NADH
ribose-5-phosphate (nucleic acids)
what is needed to make glutathione
NADPH
what is glutathione used for
repairs oxidatively damaged rbc membranes
explain the process which glutathione breaks down organic hydroperoxides
1- glutathione peroxidase breaks down/reduces organic hydroperoxides
2- produces oxidised glutathione (glutathione reductase), water and an alcohol
3- glutathione reductase is then reduced by NADPH into reduced glutathione
4- returns back to glutathione peroxidase
why does oxidised glutathione have to be broken down
can oxidise lipids
why are RBC dependent on G6PDH?
needs it to make NADPH as has no mitochondria
what metabolises the pro drug 6-MP and what is produces
- TPMT/ Thiopurine methyl transferase
- 6-methyl MP
what does the activated pro drug 6-MP cause
cell death due to blockade of DNA synthesis
what is the pro drug 6-MP used in
cancer
what are the 4 main ways we control enzyme activity?
- Inhibition (reversible or irreversible)
- Feedback regulation
- Covalent modification-post translational modification
- Proteolytic activation
what are the enzymes called that phosphorylate other enzymes
protein kinases
which AA are phosphorylated most common
seriene
thr
tyr
what do phosphatase do?
remove phosphate groups from enzymes
what does phosphorylation and dephosphorlyation cause
conformational changes in enzymes which decrease or increase their activity
what is proteolytic activation?
Irreversible activation of enzyme after removal of part of peptide chain
what is important in proteolytic activation and why
tight control by natural inhibitors (eg. antitrypsin and antithrombin III) so enzymes don’t become active in the wrong place at the wrong time
what is chymotrypsin
a serine protease: digestive enzyme
what is the structure of chymotrypsin?
3 polypeptide chains in active form- all linked by disulphide bonds
(Zymogen precursor is a single polypeptide)
what AA are in the active site of chymotrypsin
aspartate
histamine
serine
highlight the steps in the activation of chymotrypsin
1- inactive zymogen chymotrypsinogen activated by trypsin through cleavage of peptide bonds- proteolysis
2- chymotrypsinogen then undergoes further proteolysis to further activate itself into fully active form a-chymotrypsinogen
what is the precursor for trypsin
trypsinogen
how is trypsinogen converted to trypsin
1- removal of 1-6 AA in trypsinogen
2- by enteropeptidase (duodenal enzyme)
what are the most common enzymes in blood clotting cascade ?
serine proteases
what does antithrombin III bind to
thrombin
serine proteases
what does plasmin do
lysis of blood clots
highlight the steps from which a blood clot is dissolved
-TPA (tissue type plasminogen activator) binds to plasminogen
2-serine protease activity of TPA cleaves plasminogen to activated plasmin
3- Plasmin digests fibrin clot to small peptides = dissolves blood clot
when we measure for enzyme activity do we use the serum or the whole blood, and why?
serum- has no blood clotting
how can tissue specific enzymes be used as markers
if they are found in serum = indicates damage = shouldn’t be there
what are enzyme isoforms ?
enzymes with similar function, and sometimes similar amino acid sequence, but expressed from different genes
how can Differential tissue distribution of enzyme isoforms be used as markers
Some isoforms more abundant in particular point in blood- can trace to see where damage is
in what condition are enzymes amylase and lipase used as markers? and why do we use the 2 enzymes together
acute pancreatitis
- amylase gets degraded quickly
in what condition are enzymes alkaline phosphatase used as markers?
liver disease
osteoblastic bone disease
in what condition are enzymes glutamate-oxaloactetate transaminase (GOT) used as markers?
myocardial infarction
liver cell damage
in what condition are enzymes glutamate-pyruvate transaminase (GPT) used as markers?
liver cell damage
in what condition are enzymes Creatine kinase (CK) used as markers?
crush injuries
why are the times you do enzyme tests important?
different enzymes have different half-life in blood (for example: in acute pancreatitis, lipase remains elevated for longer than amylase)
what is the standard measure of enzyme activity
International Unit (IU)
what is the IU
amount of activity that will convert 1 micromole (µmole, or 10-6 moles) of substrate per minute under standard defined conditions, in specific temp, place, certain buffers
what are the products of the reaction when GPT catalyses glutamate + pyruvate ?
alanine + a-ketoglutarate
what are the products of the reaction when GOT catalyses glutamate + oxaloacetate ?
aspartate + a-ketoglutarate
what is jaundice in liver damage due to
high levels of bilirubin
what 3 enzymes increase in levels due to liver damage
GPT,GOT,BILIRUBIN
what 6 things are measured in a liver function test
- albumin
- GOT
- GPT
- total transaminase (GOT+GPT)
- alkaline phospahtase (ALP)
- direct and total bilirubin
what do low levels of albumin suggest?
general loss of secretory function/ kidney disease
what do elevated levels of GOT suggest
acute liver damage, but could originate from red blood cells or muscle, so not liver-specific
what do elevated levels of GPT suggest
more specific indicator of liver damage - high levels rules out muscle damage
what do high levels of total transaminase but normal ALP suggest?
liver necrosis
what do elevated levels of ALP suggest?
large bile duct obstruction (Caution: also high in Paget’s Disease of Bone, growing kids and 3rd trimester pregnancy)
if direct and total bilirubin levels are normal but we are still presented with jaundice what does this suggest?
problem upstream of the liver (some form of haemorrhage causing high total bilirubin)
what do elevated direct bilirubin suggest?
normal conjugation function in the liver, but failure to excrete (bile duct obstruction)
what test other than transaminases is sensitive to minor changes in liver function?
Gamma glutaryl transpeptidase (GGT)
what does slow Prothrombin time (INR) suggest?
indicates failure of the liver to synthesise and secrete blood clotting proteins such as prothrombin
what is INR/prothrombin time used to monitor?
- livers ability to produce blood clotting proteins
- warfarin usage
- Vitamin K status
what are the 3 markers of cardiac injury?
- CK (in MB form)
- Cardiac troponin-I (cTnI)
- lactate dehydrogonase
what reaction does CK catalyse
creatine + ATP —-(CK)—> Creatine phosphate + ADP
what is Creatine phosphate
sore of high energy phosphate groups in muscles
what 2 polypeptides isoforms is CK made from
M and B
what CK isoform is predominant in skeletal muscle?
-MM
what causes increased levels of MM isoform of CK
AMI(myocardial infarction) but also with skeletal muscle injury, intensive training, thyroid deficiency
what CK isoform is predominant in myocardium ?
MB
what causes increased levels of MB isoform of CK
AMI
what CK isoform is predominant in brain ?
BB
what causes increased levels of BB isoform of CK
neurological disorders, some cancers
why is CK levels measured in crush injuries?
determine severity of muscle tissue breakdown
what’s is rhabdomyolysis
muscle tissue breakdown
how can rhabdomyolysis lead to brown urine
- Muscle tissue breakdown with the release of intracellular contents, Myoglobin, into circulation
- Elevated CK levels
- Dark reddish brown urine due to Mb
why can Rhabdomyolysis be dangerous
-Mb may occlude structures of the kidney and break down into toxic compounds leading to acute tubular necrosis or acute renal failure
what can paracetamol poisoning lead to?
liver damage
up to what time can one gain full recovery from paracetamol poisoning? what happens after this tie?
12hrs
after 12hrs = irreversible liver damage - transplant?
what is pracetomal poisoning treated with
N-acetylcysteine/methionine
what does paracetamol inhibit?
the COX’s
cycloxygenase-1 and 2
what’s do COX’s activate?
prostaglandins which cause inflammation
highlight the steps in the breakdown of paracetamol
1- some paracetamol leaves first by real excretion
2- cytochrome P450 breaks paracetamol down to Quinone derivative
3- Quinone derivative then broken down by glutathione 4- excretion
biochemically, what happens in paracetamol poisoning?
too much paracetamol = too much Quinone derivative = insufficient glutathione to break it all down = toxicity
how does N -acetlycysteine work
same mechanism at glutathione : breaks down Quinone derivative
does hypo or hyper glycemic cause long term damage ?
hyper
hypoglycaemia= immediate danger
how does hyperglycaemia cause long term damage
damage tissues with microvasculature (portion of the circulatory system composed of the smallest vessels), eg. Heart
what is the significance of asparagine in cancer cells?
Tumor cells deficient in Asn synthetase, therefore Asn essential
how do tumour cells obtain their asparagine
take it from healthy cells, diet
is asparagine essential to normal healthy cells?
no
how can we use asparaginase to treat childhood acute lymphoblastic leukaemia
Asparaginase can convert asparagine to aspartate and starve tumour = stop them growing
meanwhile normal cells unaffected
what is urease used for
in kidney dialysis to remove urea
what does urease break urea down into
2NH3 (removed by carbon) and CO2 (returned to lungs)
