1.3 - Enzyme Kinetics III: Enzyme Modulation and Examples Flashcards
regulation
key characteristic of enzyme is it’s ability to be regulated
what factors can regulate enzyme activity? (2)
- physical changes within cell (i.e. temp/pH)
- gene expression changes can also regulate enzymatic activity
mechanisms involved in regulating enzymatic function (4)
- co-factors
- inhibitors
- allosteric regulators
- covalent modifications
allosteric regulation
involves binding of a regulatory molecule to the enzyme at separate site to active site (allosteric site)
possible effects of allosteric regulation (2)
- inhibitory
- enzyme activation
what happens when the allosteric regulator binds to an enzyme?
can induce conformational changes to induce or inhibit enzymatic activity
cooperativity
form of allosteric regulation that can enhance enzyme activity
cooperativity mechanism
often involves binding of substrate to one of enzyme subunit’s active site, locking it in active conformation
example of cooperativity (not enzyme)
haemoglobin - oxygen binding to one subunit enhances affinity for oxygen to other 3 active sites (positive cooperative binding)
feedback inhibition
product of a reaction can inhibit the reaction pathway
where does feedback inhibition often occur?
metabolic processes to prevent wasting resources or toxic levels of certain products
example of feedback inhibition (breakdown of threonine by threonine deaminase) (3)
- TD has 2 allosteric sites: high/low affinity site
- isoleucine (pathway end product) binds at high affinity site
- increases binding of isoleucine at low affinity site, deactivating enzyme by inducing inactive state
covalent modifications
enzymes can be modified by various post-translational modifications
types of covalent modifications (3)
- phosphorylation
- methylation
- ubiquitination
what does glycogen phosphorylase catalyse
phosphorolysis of glycogen to glucose-1-phosphate
glycogen phosphorylase forms (2)
- unphosphorylated enzyme (b)
- phosphorylates form (α)
what allosterically regulates phosphorylase b? (3)
- AMP
- ATP
- G6P (product of the reaction)
what form of phosphorylase b does AMP promote?
active form
what form of phosphorylase b do ATP and G6P?
inactive form
what allosterically regulates phosphorylase α?
glucose - promotes inactive form
glycolysis pathway
converts glucose to pyruvate, producing ATP and NADH (sequential, 10 enzyme reaction)
how is glycolysis heavily regulated at each step? (3)
- gene expression levels
- allosteric inhibition
- post-translational modifications
key regulatory enzymes in glycolysis (3)
- hexokinase
- phosphofructokinase
- pyruvate kinases
how is hexokinase inhibited? (glycolysis)
by G6P, the product of its reaction, via allostery
how is phosphofructokinase activated/inhibited (2)
- activated - AMP
- inhibited - ATP and citrate
last step of glycolysis pathway
pyruvate kinase converts phosphoenolpyruvate into pyruvate
how is the conversion of phosphoenolpyruvate into pyruvate by pyruvate kinase (PK) regulated by allostery? (2)
- fructose-1,6-biphosphate binding alters active site of PK, enhancing activity
- PK also allosterically inhibited by ATP, long chain fatty acids and alanine
(most sensitive regulation of PK)
how is pyruvate kinase (PK) activated?
by covalent modifications, protein kinase A can phosphorylate PK in response to hormone glucagon and epinephrine
components of cell signalling (3)
- signal
- receptor
- effector
signal cascades
amplifiers of initial signal (cell signalling)
form of signals
most signals are chemical in nature, can be driven via changes in voltage, pressure or pH
receptor tyrosine kinases (RTKs)
major class of enzymes that function as receptors to transmit signals
kinases
enzymes that catalyse the phosphorylation of proteins
result of RTKs on cell surface binding to their corresponding ligand
dimerization of the RTK and autophosphorylation of the cytoplasmic tails of the receptor (RTK now active and can phosphorylate other proteins)
SH2 domain containing proteins
can be other kinases, phosphatases or other important signalling proteins
ligand-RTK binding signalling cascade
induces gene expression changes, differ depending on the ligand RTK involved
example of ligand binding to RTK signalling cascade (10)
- growth factor (e.g. EGF) binds to an RTK (EGFR), inducing:
- dimerisation and autophosphorylation of RKT cytoplasmic tail
- SH2 domain containing proteins such as Grb2 can bind to pY residues
- binds to adaptor protein SOS
- SOS can activate RAS by exchanging RAS-bound GDP for GTP
- active RAS then starts enzymatic cascade by binding to Raf
- Raf, a Ser/Thr kinase, then phosphorylates MEK
- MEK, a Ser/Thr kinase, phosphorylates MAPK/ERK, a Ser/Thr kinase
9 MAPK/ERK translocates to nucleus - phosphorylating transcription factors such as Fos, Jun and Myc
