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
