SfM - Enzymes Flashcards
what is an enzyme?
- globular protein
- biological catalysts (increases reaction rate)
what is a co-factor?
non-protein component needed for activity
what is a co-enzyme?
complex organic morecule
what is meant by a prosthetic group?
non-protein co-factor bond to the enzyme i.e Haem group
what is the difference between an apo/holo-enzyme?
- apoenzyme = is the enzyme in its inactivated state i.e protein w/o co-factor
- holoenzyme = the enzyme in its functional/active state with co-factor attached
what is a substrate?
- molecule that is acted upon by enzyme
what is an active site?
- part of the enzyme that the substrate binds and is acted upon
what is the function of enzymes?
- increase rates of spontaneous reactions
- lower the activation energy of biological reactions
- accelerate movement towards reaction equilibrium
how do enzymes reduce activation energy?
- enzymes force the substrates to be correctly orientated by binding them in correct formation needed for reaction to go
- conformational changes occur in the protein structure when substrate binds
what is meant by enzyme kinetics?
- study of chemical reactions between enzyme and substrate
- More substrate you put in, the quicker the initial reaction velocity/speed (V0). Initially the reaction kinetics will produce a linear response as there is large amounts of enzyme and substrate – however, this will eventually plateau as substrate conc. is reduced and enzymes are saturated (active sites)
what is the rate limiting step of enzyme kinetics?
ES –> E + P
- reaction must be proportional to amount ES
what is Km?
the ratio of rate constant for:
breakdown of ES to E+S compared to
formation of ES from E+S
- indicates affinity of the enzyme for it’s substrate
what is Vmax?
- tells you how fast a reaction is proceeding when the enzyme is saturated with substrate
what does a high Km and high Vmax mean?
- lower affinity
- requires greater conc to reach max
(glucokinase - liver)
what does low Km and low Vmax mean?
- high affinity
- requires less to reach max
(hexokinase - body)
how do glucokinase/hexokinase work?
- blood glucose increases
- glucokinase activity increases (liver hit with high conc of glucose)
- hexokinase does not respond as it is continually working at Vmax (doesn’t take unnecessary glucose)
what is an isoenzyme?
- different enzymes that catalyse the same reaction (variable rates of reaction)
- products of different genes
- can be separated via electrophoresis
what does an ordered sequential reaction mean?
- there is specific order of binding and release of substrates of the reaction and then products
what does a random sequential reaction mean?
- it doesn’t matter if the substrate or the co-factor bind to enzyme first or what product is released first
what is an allosteric enzyme?
- large multi-subunit enzymes which contain many active sites
(can host many reactions at the same time - either same or different)
what will affect an enzyme?
- temperature (increased temp = increase reaction rate. NOTE - could denature enzyme eventually)
- pH (pH changes alter charge of amino acids - if active site aa charge is altered - enzyme ceases to function)
what kind of enzyme inhibitors are there?
- competitive inhibitor
(enzymes non-covalently bind & compete w/ substrate for active site - usually resemble substrate) - uncompetitive inhibitor
(bind to enzyme and affect reaction process w/o binding to active site) - non-competitive inhibitor
(non-covalently bind to enzyme but not on active site)
what happens when non-/competitive inhibitors are present?
- presence of CI leads to a decrease in affinity to the substrate, so Km increases, however increasing substrate concentration can overcome this inhibition, so same Vmax
- presence of n-CI, substrate is still able to bind to active site so Km stays same but Vmax decreases
what is a transition state analogue?
- as maximal interaction occurs between enzyme and the transition state rather than substrate, an analogue that mimics the transition state is created instead
what is a catalytic antibody?
- an antibody that is specific to a transition state molecule
what is an irreversible inhibitor?
- covalently bind to the enzyme, meaning it cannot be removed (cyanide)
how are enzymes controlled?
- feedback inhibition - some pathways prevent build-up of the end-product but turning off the enzyme when product conc gets too high
what are allosteric effectors?
- cell metabolites that bind non-covalently to a site that is not the active-site, this changes enzyme structure
- can be activators or inhibitors (increase/decrease likelihood of binding)
describe stages in concerted model
- with no substrate the enzyme flips between open and closed conformation
- when one molecule of substrate binds it locks other binding sites in open state, stopping them from flipping, allows more S to bind
- allosteric activators - stabilise open conformation
- allosteric inhibitors - favour closed formation
describe stages in sequential model
- substrate binding causes a change in one subunit
- this causes a change in another sub-unit allowing to bind S more readily and so on…
- there is sequential increase of affinity to substrate
in what ways can enzymes be regulated?
- covalent modification i.e phosphorylation/proteolytic cleavage
- multiple phosphorylation sites allow fine control of enzyme - not on/off more of a dimmer switch
- proteolytic cleavage - inactive precursor protein can be cleaved by proteases to give active form
