Enzymology Flashcards
Factors affecting enzymes
pH, heat, type of buffer, conc of enzyme or substrate or cofactor
Different phases of enzymatic reactions
Lag phase, true inital rate,
What is the steady state transition state theory?
Enzymes lower activation energy to increase rate, but has no effect on position of equilibrium (where activated and reactants are equal)
- The standard free energy of activation describes the amount of energy necessary to reach transition state
Describe two rate measurement methods
Fixed time method: enzymes with low Km (high affinity)
- measure conc of substrate and product at two points
Continuous monitoring method: use enzymes with low affinities (high Km)
- monitored continuously
Example of enzyme used as reagent
specific bio-reagents
Glucose oxidase: react glucose with oxygen to produce H2O2. H2O2 is then used in a seperate reaction with peroxidase to produce a coloured compound (oxidised o-toluidine).
Other example when coupled enzyme provide specificity.
- Lack of specificity in primary enzyme (hexokinase) is compensated by a high specificity secondary enzyme (G-6-Pi-DH)
Describe an end point assay.
Where reaction equilibrium is unfavorable to the desired direction. A trapping reagent may be used to pull the equilibrium to desired way.
E.g. Lactate analysis.
Firstly converted from lactate to pyruvate + NADH using LDH, and a second reaction (trapper) pyruvate + hydrazine produce its hydrazone. Here NADH is measure and the second reaction draws the equilibrium towards hydrazine reaction
Enzymes as labels in immunoassays
- Often analyses very low conc of analyte. Can ampligy the signal as long as they have appropriate substrate
- enzymes with high Kcat (turnover) produce more signal
Two techniques of enzymes as labels in immunoassay
EMIT
ELISA
Measurement of enzymes effect on signal produced
- Potential ability to produce large signal despite its small conc
Why use enzymology in a clinical setting?
- Detect suspected disease at pre-clincial stage
- Confirmation of suspected disease and assess severity
- Localisation of organ pathology
- assessing response to therapy
- Characterisation of organ pathology
- Organ function assessment
- Assessing genetic susceptibility to drug side effects
Define isoenzyme
Multiple forms of an enzyme that possess the ability to catalyse the same reaction even with differing amino acid sequences. Display different kinetic parameters, such as regulatory properties
Example of an isoenzyme
Creatinine kinase has a dimeric structure of M (muscle) and B (brain) subunits.
- in muscle tissue CK M-M
- in brain tissue CK B-B
- in cardiac CK B-M
- All forms have the same enzymatic activity
Describe the isoenzymes of alkaline phosphatase
- Tissue nonspecific - gene on short arm of chromosome 1.
- Differential glycosylation - tissue specific isoforms e.g. liver and bone
Whilst there are genes on chromosome 2 producing other tissue specific isoenzymes - intestinal, placental and germ-cell
What are some distinguishing features of alkaline phosphatase which mediate the identification of each isoenzyme in clinical setting?
Heat
- 56C bone ALP is more heat labile than liver
- 65C all except placental ALP are rapidly inactivated
Sialic acid content
- Incubation with neuraminidase retards electrophoretic migration of all ALP except intestinal
- Wheat germ agglutinin binds preferably to bone ALP
Immunoassay
- Bone ALP monoclonal antibodies available
How can serum cholinesterase (CE) be used to assess drug side effect susceptibility?
Used to assess suxamethonium and mivacurium sensitivity. These are used in anaesthetic procedures.
- Certain CE phenotypes cant degrade the drug efficiently and is at risk for apnoea
- FF, FS and AF intermediate sensitivity to suxamethonium
- AA, AS, SS experience prolonged paralysis
- UA or UF carry small risk
What is the use of thiopurine methyl transferase (TPMT) in diagnostic enzymology?
Thiopurine drugs are used to treat inflammatory and autoimmune disease. Drug catabolised to inactive metabolites by TMPT. Active to 6TGN.
- Measurement of TMPT is recommendend before tiopurine drug treatment. To ensure people with little to no TPMT activity dont take thiopurine drugs and get severe side effects
Should a patient with low activity of TPMT take thiopurine drugs?
No, will experience adverse side effects such as myelosuppression (6-TG)
Should a patient with high activity of TPMT take a higher dose of thiopurine drugs?
An increased dose may lead to accumulation of inactive metbaolites and increase risk of hepatotoxicity (6-MMP)
Diagnostic enzymology to investigate hypertension
Using plasma renin activity assay. Renin catalysed conversion of endogenous plasma angiotensinogen to angiotensin I (measured by MS)
Angiotensin-converting enzyme can be used to investigate what disease?
Sarcoidosis
Neurone specific enolase converting enzyme can be used to investigate what disease?
Tumour marker, raised in neuroblastoma, carcinoid, melanoma, and small cell lung cancer
How is enzymatic activity calculated?
Beer lambert law is modified to produce a viable formula.
Enz. Act. = abschange/minx106xtot. vol/Exlxsample vol
Michaelis Menten
v=Vmax(S)/Km+(S)
How does competitive, non-competive, uncompetitive inhibitors affect the inverse binding curve?
Non-comp: Km stays same but y-interecept increase (Vmax decrease)
Competitive - Km increases and Vmax is the same
Uncompetitive - decrease in Vmax and Km
Continuous vs fixed method for rate measurement.
Continuous assess reaction progress, ensures true initial rate measurement obtained and identify any lag phase
What is the unit for enzymatic activity?
SI unit is katal
- amount of enzyme converting 1 mole of substrate in 1 sec
International unit -U
- amount of enzyme which convert 1umole of sub per min
1U/L = 16.67 nkatal/L
What are some more common examples of enzymes as reagents?
Urate, triglycerides, ammonia, creatinine, lactate, cholesterol and urea
How has the Jaffe method of creatinine measurement been improved?
Jaffe method uses alkaline picrate as colour reagent, but this method is prone to interferences by bilirubin.
- So modern analysers use Jaffe, but when high bilirubin is detected it also uses enzymatic creatinine analysis
Factors determining enzymatic activities in serum/plasma?
Age, gender, pregnancy, genetics, drugs, disease process, treatment e.g. surgery
