Enzymes Flashcards
What is an enzyme
Protien that catalyses chemical reactions without itself being destroyed/altered - seek to lower the Ea
What is different with enzyme catalysed reactions compared to chemical catlysts
Can be regulated
Subvstrate
Reactant in catalysed reaction
Active site
Part of enxyme that binds to substrate to form E-S complex
product
Substance produced by enzyme-catalysed conversion of substrate
Cofactor
non-protien component needed for the reaction (e.g. magnesium)
Coenzyme
Heat-stable substance which can aid enzyme reactions
Isoenzyme
enzymes that catalyse the same reaction but vary in structure and other biochemical properties
Activation energy
Energy needed for the reaction to proceed
How do enzymes inc the rate of reaction
by lowering the Ea
How do enxymes lower the Ea (3)
- Entropy reduction - Enzymes force the substate to be correctly orientated by binding them in the formation they need to be in for the reaction to proceed
- Desolvation - weak bonds between S and E essentially replace most or all of the H-bonds between substrate and aqueous solution
- Induced fit - conformational changes occur in the protien structure where the substrate binds
basic enzyme reaction
- substrate enters active site of enzyme
- forms an enzyme-substrate complex, enzyme changes shape slightly as substrate binds (induced fit)
- forms an enzyme-product complex
- products leave active site of enzyme
Michaelis-Menton plot
V0= Vmax [S]/Km
V0= initial reaction velocity
Vmax= maximum reaction velocity
[S]= substrate concentration
Km= the substrate concentration when the reaction is at ½ the maximum velocity (Vmax)
Michaelis-menton plot diagram
look one up
What does Km measure
Specificity of enzyme for substrate
What does a low/high Km value mean?
- Low = good fit
- High = poor fit (takes lots of substrate to get to 1/2Vmax)
What does Vmax measure
how fast a reaction is proceeding when the enzyme is saturated with substrate
What can affect enzyme reactions
- Enzyme concentration - inc, inc rate
- Substrate concentration
- Temp
- pH
- Inhibitors (competitive/non-competitive)
What are competitive inhibitors
Inhibitor binds to AC of enzyme and prevents the substrate from binding
What happens to Vmax and Km with competitive inhibition
- Vmax = unchanged
- Km = inc because it takes more substrate to overcome the inhibition
What is non-competitive inhibiton
Inhibitor binds to a secondaty site on the enzyme which changes the shape of the AS and prevents the substrate from binding
What happens to Vmax/Km during non-competitive inhibition
- Vmax = decreased
- Km = remains the same
Why measure enzymes
- Detection of suspected disease at pre-clinical stage
- Confirmation of suspected disease and assessing severity
- Localisation of disease to organs
- Characterisation of organ pathology
- Assessing the response to therapy
- Organ function assessment
- Assessing genetic susceptibility to drug side effects
- Detection of inherited metabolic disease
- Detection of vitamin deficiencies**
Factors to determine enzyme activity in serum/plasma
- age
- gender
- pregnancy
- race
- time of day
- genetics
- drugs
- disease process, progress and treatment e.g. surgery
Serum enzyme concentrations
Although there are some enzymes secreted from cells, serum E conc generally Low and only rise when there is damahe to cells and release of contents
what is the release of enzymes from cells triggered by - factors to determine E activity in serum/plasma
- hypoxia
- cellular damage
- physical damage
- immune disorders
- microbiological agents
- genetic defects
- nutritional disorders
hypoxia
Loss of O2 supply due to occlusion, inadequate oxygenation, or O2 carrying capacity
What could cause cellular damage
chemicals/drugs
What could physical damage include
trauma, surgery, burns, radiation…
Give some examples of immune disorders
anaphylaxis, automimmune diseases
What could microbiological agents include
bacteria, viruses, fungi, protozoa, helminthhs
WHat could nutritional disorders include
protien-calorie, vitiman, mineral deficiencies
How do we analyse enzymes in the lab
- Rate of an enzyme-catalysed reaction is directly proportional to the amount of enzyme present
- Progress of conversion of the substrate to product is monitored:
Fixed-time - at an end-point
Continuous - throughout - Often measurement of an end product using spectrophotometry - coloured end product
- Electrophoresis - to determine type (origin)
What are some problems with enzyme measurement
- Not specific i.e. can be found in more than one tissue in the body.
- Particular requirements - temperature, pH etc.
- Assays must be optimised to conditions E require
In what clinical situations are enzymes used - clinical diagnosis
- tissue damage
- Determine origin of affected tissue
- Disease related to enzyme defects
What are some facts/conditions of enzyme use clinically
- often not specific
- need particular conditions for measurement
Why is enzyme activity measured in a clinical setting
enzyme assays are valuable for diagnosis of several diseases in clinical settings due to their high sensitivity, specificity, and rapid response.
what factors can influence enzyme activity in samples
temperature, pH, enzyme concentration, substrate concentration, and the presence
Michaelis constant definition
the concentration of substrate that is transported at half the maximal velocity (Vmax) of transport; a measure of the affinity of the transporter for its substrate.
WHat does knowledge of the michaelis constant allow to be deduced about the nature of the catlysed reaction
A measure of how well a substrate complexes with a given enzyme, otherwise known as its binding affinity.
