Regulation - keeping the system in balance Flashcards
Describe cell factory regulation
- regulation by mass action
- feedback loops & allosteric regulation
- regulation by spatial organisation of enzymes
- regulation by post-translational modification of enzymes
Describe regulation by gene expression
changing the amounts of enzymes present
Give an example of regulation by gene expression
- lacZ encodes beta-galactosidase
- lacY encodes beta-galactoside permease
- lacA encodes LacA, an
enzyme that transfers an acetyl group from acetyl- CoA to β-galactosides
Describe beta-galactosidase permeate
transmembrane transporter that pumps lactose into the cell
LacA
β-galactoside transacetylase
Describe feedback regulation of ATCase
- rate of the reaction catalysed by ATCase is fast at low concentrations of CTP
- slows as CTP concentration increases
- ATCase is inhibited by CTP
ATCase
aspartate transcarbamoylase
Describe the Carbamoyl phosphate pathway
- Carbamoyl phosphate + aspartate -> N-Carbomoylaspartate via ATCase
- Pi -> CTP
CTP
cytidine triphosphate
Describe ATCase kinetics
- not Michaelis-Menten
- sigmoidal
- very large change in activity within a narrow substrate concentration range
Describe ATCase
- consists of separate C and R subunits
- 6 catalytic subunits arranged as a pair of trimers, one sitting on top of the other
- 6 regulatory subunits arranged as three dimers that sit between the catalytic subunit junctions
- CTP binds to a site on each regulatory subunit
C
catalytic
R
regulatory
Describe ATCase active sites
- each catalytic trimer has three active sites at the junction between catalytic subunits
- contains key amino acid residues from each of the two catalytic subunits that make it up
Substrate binding and inhibitor binding
favour different conformational states
Describe the R state
- catalytic subunit rotation brings active site residues closer together: more efficient catalysis
- catalytic triads are 1.2 nm further apart, favours substrate binding
- more active
- favoured by substrate binding
Describe the T state
- less active
- favoured by CTP binding
Describe the sigmoidal kinetics of ACTase
result of the combined kinetics of the T and R states of the enzyme
Describe allosteric activation & inhibition of a single enzyme
solves the energy vs carbon skeleton partitioning problem in glycolysis
Metabolic pathways typically contain
multiple feedback and feedforward loops to provide fine regulation of pathway flux in response to varying demands from the rest of the metabolic system
Describe the biosynthesis IMP
- 10 steps
- catalysed by 6 enzymes
- organisation of enzymes into a cluster accelerates pathway flux
IMP
- inosine monophosphate
- precursor for the synthesis of purines
purines
adenine & guanine
Describe enzyme clustering
- faster because of increased local concentration of enzyme
- prevents other pathways from competing for intermediates
Why is phosphorylation an effective means of regulation?
- phosphoryl group adds two negative charges to the protein and can form 3 or more hydrogen bonds
- can take place in less than a second or over a span of hours
- produces highly amplified effects. One kinase can phosphorylate hundreds of target proteins in a short interval
- uses ATP as the phosphoryl donor and thus it is linked to the cell’s energy status
Describe the addition of a phosphoryl group
alters the structural conformation of the enzyme and affect substrate binding and catalytic activity
Describe phosphorylation and dephosphorylation
controlled by kinase and phosphatase enzymes
