Paper

Question 1

what does this paper look at

Answer 1

ATCase from ecoli

*Ecoli has almsot identical to human form of this enzyme

Question 2

Questions to be able to answer

Answer 2

What, specifically, is the D236Ac mutant?
What do we learn from Figure 1?
What do we learn from Figure 2?
What is the significance of the D236Ac mutant?

What does the structure of the D236Ac mutant bound with CP bound tell us about how ATCase functions?

Question 3

What is the events in the ATCase reaction

Answer 3

Events in the ATCase reaction occur in a fixed order:

1) Carbamoyl phosphate (CP) binds the active site
2) Aspartate then binds the active site - this leads to large changes in quaternary structure
3) Condensation reaction occurs
4) The product N-carbamoyl-L-aspartate (CA) is released

5) Pi is released last

• As long as substrate is available, the enzyme remains in the active R state

*why not just keep adding substrate? analysis requires sample to be in machine for long time, wont be able to keep adding in substrate the volume adn density will inc which might interfere

Question 4

what is the key residue pair

Answer 4

-Two charged amino acids

• Asp236 on the catalytic chain (D236c) *236th aa in peptide on catalytic chain
• Lys143 on the regulatory chain (K143r)
• interact between chains to stabilize the T state
• After mutation of either of these residues to alanine (D236Ac or K143Ar), the enzyme can no longer adopt the T state

*these are artificially induced

• In the absence of ligands, WT and D236Ac ATCase have different structures (wt likes to sit in T state and mutation likes to sit in the R)
• However, in the presence of PALA, WT ATCase and D236Ac are the same (bc pala shifts wild type to the R state)

Question 5

what occurs in the absense of negatively charged R group of D236 or positively charged R group of K143

Answer 5

ion pair the stabilizes the T state is eliminated

Question 6

what are the substrate/ substrate analog compounds:

Answer 6

Carbamoyl phosphate (CP) - substrate

• Phosphonoacetamide (PAM) = non-reactive analog of CP
• malonate = non-reactive analog of Asp

Question 7

exaplain

Answer 7

• wt: when only CP is bound or no ligand have same shape, T state

D236Ac: no ligand has a different shape, its in between the T and R then just binding one substrate pushes it all the way to the R state, see it is same shape as PALA

Summary

• in the absence of ligand, WT ATCase and D236Ac are different
• bound to PALA, WT and D236Ac are very similar - CP has little effect on WT ATCase
• CP mimics effect of PALA in D236Ac

Question 9

Answer 9

*PAM is an analog of CP (substrate)

• when bound to PAM D236Ac adopts the R state, note in WT is not in R state configuration

Question 10

Answer 10

comparison of the catalytic chains

• show super imposed structures c is C1 of WT ATPase (PAM) overlapped with C1 of D236Ac (PAM)
• C1 of WT ATCase (PAM + malonate_ overlapped with C1 of D236Ac (PAM) see how they are basicaly yhe same

Question 11

what is the outcome of Asp236Ac mutant

Answer 11

eliminates ion pair formed between Asp 236c and Lys 143r which typically stabilizes the T state

• in absence of Asp236c–Lys143r interaction (which connects the c and r subunits) CP can push ATCase into the R state
• direct interactions between the c and r subunits regulate the conformation of the enzyme by stabilizing the T state

**net result is mutant never adopts the low activity T state, always in patially active conformation