Enzymes 2 Flashcards
what reaction do both glucokinase and hexokinase catalyse?
Glucose +ATP–> G6P + ADP
phosphorylation of glucose
why is glucose converted into G6P?
so glucose is “trapped” inside cells since G6P cannot be moved out of the cell
what is the Km and Vmax for glucokinase?
High Km
High Vmax
what is the Km an Vmax for hexokinase?
Low Km
Low Vmax
therefore, what is the glucokinase affinity for glucose?
Low affinity for glucose
therefore what is the hexokinase affinity for glucose?
High affinity for glucose
where is hexokinase found?
In RBCs (erythrocytes); which is why it’s constantly working since they don’t have mithchondria and need to generate energy
How is G6P formed?
ATP transfers phosphate group to glucose to make G6P. G6P is the metabolised in glycolysis which generates energy to form ATP or is converted to glycogen for storage.
when is hexokinase active?
all the time; stays relatively constant (can effectively use low levels of glucose but is quickly saturate)
- allows works at its Vmax
what can inhibit hexokinase?
G6P (if there’s lots)
where is glucokinase found?
in the pancreas (by Beta cells) and liver (by parenchymal cells)
when is glucokinase active?
- only when intracellular conc. of glucose in the hepatocyte is elevated/ increased (when a carbohydrate rich meal is eaten)
- when there is a large influx of glucose (doesn’t become saturated until very high glucose levels are reached)
can glucokinase be inhibited by G6P?
No, only hexokinase can
why is glucokinase action important?
- prevents large amounts of glucose from entering the circulation following a sugar-rich meal.
- prevent hyperglycemia
patients with diabetes mellitus show less activity of which enzyme?
glucokinase
patients with haemolytic anaemia show less activity of which enzyme?
hezokinase
what is the effect of insulin on hexokinase and glucokinase activity?
hex: not affected
glu: activated
what is the effect of carbohydrate on hexokinase and glucokinase activity?
hex: not affected
glu: activated
what is the effect of starvation on hexokinase and glucokinase activity?
hex: not affected
glu: inhibited
what 3 factors are needed for measuring enzyme activity?
- measure the initial rate
- have substrate in excess
- check that activity is proportional to enzyme concentration
what are isoenzymes?
different enzymes that catalyse the SAME reaction (e.g. hexokinase and glucokinase)
how can isoenzymes be studied?
by electrophoresis (separating plasma proteins according to their charge and size
what are the 2 types of reactions which occur if two or more substrates are involved in a reaction during a transfer of groups?
- random order or ordered with ternary complex
2. no ternary complex formation
what is the ternary complex?
ES1S2 (enzyme combined with the 2 substrates)
what happens in an ordered sequential reaction mechanism?
the enzyme exists in a ternary complex, first with the substrates of the reaction and then with the products of the reaction (there is order to it, step after step)
what happens in a random sequential reaction mechanism?
the order of binding and release of substrate and product is random but formation of ternary complex still occurs first with substrates and then with products of the reaction.
what happens in a no ternary complex sequential reaction mechanism?
- substrates bounce on and off the enzyme as they are catalysed (e.g. conversion of amino acids)
what is cooperative binding?
one substrate binding to one enzyme subunit can cause changes in other active sites and affect their binding affinity on other subunits (binding of one thing triggers protein structure to change shape allowing better binding in other areas)
what does the kinetic graph curve for allosteric enzymes look like?
the reaction velocity start off quite low at low substrate concentration however they gradually increase
what 3 factors can affect enzyme velocity/ action?
- Temperature
- Inhibitors
- pH
how does increase in temperature affect enzymes?
- more molecule collisions
- increases internal energy of molecules
- can lead to denaturation
how does the pH affect enzymes?
-pH changes charge of amino acids (proteins will repel each other and not stick together)
what’s a competitive inhibitor?
bind directly to the active site (mimic substrate)
what’s a non-competitive inhibitor?
binds to other areas of the enzyme that isn’t the active site by inducing a conformational change to the enzyme and preventing further substrate binding
can increasing substrate concentration overcome competitive inhibition?
yes
what bond is made by a competitive inhibitor use to bind to the active site?
non-covalent
what effect does a competitive inhibitor have on the Km and Vmax?
Increases Km (lowers affinity for substrate) Vmax remains the same
what is AZT used to treat?
HIV
how does AZT work?
mimics the ordinary DNA precursor thymidine triphosphate (TTP)
what are transition state analogues?
- chemical compounds with resemble the transition states of a substrate in an enzyme-catalysed chemical reaction
- maximal interaction occurs between enzyme and transition state rather than the substrate
what is the free energy of a transition state?
highest free energy (and not very stable)
what is an example of a transition state analogue?
Oseltamivit (Tamiflu) (prevents spread of viral infections)
what are catalytic antibodies?
- antibodies which are specific to transition state molecules can be made
- antibodies specific to enzyme active sites could be made
why are transition states extremely difficult to isolate?
they are intermediate steps between an enzyme-substrate complex and an enzyme-product complex
what disease produces a naturally occurring catalytic antibody?
lupus erythematosus (antibodies attack connective tissue on joints, skin, kidneys, heart and lungs)
how is the Km and Vmax affected by the non-competitive inhibitors?
Km remains unchanged
Vmax will decrease
what are irreversible inhibitors?
type of inhibitors which bind to the enzyme covalently and therefore irreversibly
what are regulatory enzymes in a reaction or pathway?
enzymes which have the greatest effect on the overall pathway
which enzyme is usually the one holding the “regulatory” step?
the first enzyme in a pathway
what are the 2 ways that regulatory enzymes modulate reactions?
- allosteric enzymes
2. covalently modified enzymes
what is feedback inhibition?
a build up of an end product of a pathway or a key junction in a pathway can ultimately slow down the whole pathway (final product inhibits the pathway if the pathway if highly active)
what are allosteric effector?
- usually cell metabolites which bind non-covalently to a site on the enzyme that isn’t the active site
- changes enzyme’s structure and conformation
- some are activators and some are inhibitors
- example of a non-competitive inhibitor
what are the 2 main models explaining allosteric enzyme kinetics?
- concerted model
- sequential model
(all enzymes will fit one of the two models)
what is a concerted model?
- with no substrate present, the enzyme will flip between two conformations
- all subunits must be in the same conformation (flip in concert)
- when a molecule binds to enzyme, this “locks” other binding sites stopping the flipping allowing other molecules to bind easier
- more enzymes more sensitive to low levels of substrate
how do allosteric enzymes affect the concerted model?
- allosteric activates will stabilise the “open” conformation allowing S to bind more effectively
- allosteric inhibitors will stabilise the “closed” conformation and make it difficult for S to bind effectively
what is a sequential model?
- no flipping between different conformational states
- subunits exist in a conformation that can bind S, activators and inhibitors.
- binding causes conformational change
- binding of S sensitises the enzymes to bind more
- sequential changes to protein change
what do protein phosphatases do?
remove phosphoryl groups
what do protein kinases do?
add phosphoryl groups to proteins
why is phosphorylation (covalent modification) important?
- allow fine control of enzyme function
- enzyme has a finely tuned activity dependent on the signal it receives
what are proproteins and proenzymes?
enzymes which exist as an inactive precursor protein
what can proproteins be cleaved by to give active enzyme?
proteases
what enzymes are regulated using proproteins?
digestive enzymes
why do enzymes need to be cleaved?
they are cleaved to be used in an ACTIVE form since digestive enzymes (zymogens) cannot exist in active form all the time as they would digest tissue
in what 3 ways can enzymes be modulated?
- allosterically
- covalent modification
- proteolytic cleavage
what are 4 main enzyme inhibitors types?
- competitive inhibitor
- non-competitive inhibitor
- irreversible inhibitors
- feedback inhibitors
