Lecture 10- Protein Behavior I

Michaelis-Menten model of enzyme kinetics

-idealized model

-a significant number of enzymes behave very differently

Kinetic similarities

-exist between non-allosteric enzymes (myoglobin, chymotrypsin); hyperbolic

-exist between allosteric enzymes (hemoglobin, ATCase); sigmoidal

cooperative effects

-observed with allosteric enzymes

-accompanied by changes in quaternary structure and binding affinity of the substrates

-positive and negative cooperativity (both allosteric binding)

Positive cooperativity

-the binding of a ligand increases the affinity for the next ligand

-multiple binding sites affect each other

Negative cooperativity

-the binding of a ligand decreases the affinity for the next ligand

reaction pathways

-how many enzymes catalyze steps

-contain a series of sequential reactions

-energetically demanding

-carbamoyl phosphate + aspartate ⇌ carbamoyl aspartate + HPO₄^2-

feedback inhibition

-another way enzymes are controlled through the binding of ligands

-the final product of a metabolic pathway inhibits the first reaction in the series

-inhibition of the first step effectively shuts off the entire pathway as the concentration of the final product gets larger

the synthesis of nucleotides is…

-tightly controlled in order to mitigate overproduction of a single nucleotide (could cause a genetic mutation)

CTP

-final product of reaction pathway initiated by ATCase

-inhibits the activity of ATCase

-CTP binds allosterically to the regulatory (R) domain of ATCase and shuts down its ability to generate carbamoyl aspartate

-this effectively shuts off the production of CTP by inhibiting the first step in the biochemical pathway

Sigmoidal curve

-the graphical representation of the ATCase reaction velocity as a function of substrate concentration is a sigmoidal curve

-indicates it is an allosteric enzyme that does not follow Michaelis Menten kinetics (in contrast to non-allosteric enzymes with a hyperbolic curve)

CTP and ATP

-CTP inactivates ATCase, while ATP activates the enzyme (balance of purines and pyrimidines)

-because CTP does not change the V_max of ATCase, non-competitive inhibition cannot be the case here

-CTP is not a competitive inhibitor of ATCase either - therefore the model of enzyme inhibition becomes more complex

ATCase

-composed of 2 catalytic (C domains) trimers and 3 sets of regulatory (R domains) dimers

C domain in ATCase

-bind aspartate and carbamoyl phosphates

R domain in ATCase

-bind ATP or CTP and control the activity of the catalytic domains

Binding of ATP or CTP

-cause conformational shifts in the enzyme that affect its overall activity

-despite differences in structure, each nucleotide is thought to bind to the same site within the R domains of ATCase