biochem e2l5: enzyme regulation

chymotrypsin 8 steps according to cummins

1. general base catalysis

   1. his57 makes ser195 nucleophile

2. covalent catalysis and TS stabilization (oxyanion hole)

   1. ser195 nucleophilic attack forms tetra O- in o hole w/ Gly

3. acid catalysis

   1. tetrahedral collapse and this57 protonates LG

4. product release

5. general base catalysis

   1. his57 makes water nucleophile

6. covalent catalysis and TS stabilization (oxyanion hole)

   1. OH- nucleophilic attack forms tetra O- in o hole w/ guy

7. general acid catalysis

   1. tetra collapse his57 protonates Ser195-OH

8. product release

1. allostery

   1. homo v heterotropic, where bind?

   2. positive or neg

2. covalent modification

3. cleavage

4. neg allosteric modulator v inhibitor?

1. reversible non-covalent binding of homotropic or heterotropic modulators (activator or inhibitor)

   1. homo = multisubunit enzyme that can bind more than one substrate at a time, but binding of one alters the binding of another

      1. often bind catalytic protein active site

   2. hetero = not a substrate, modulator binds somewhere else other than active site on enzyme but modifies active site activity

      1. modulator and substrate are DIFFERENT

      2. often binds regulatory subunits

2. like phosphorylation

3. activation of zymogens (pro-enzymes, not pro-protein) by proteolytic cleavage

4. inhibitor = no activity, neg also = reduced activity

1. 
   

1. allosteric enzymes show what relationship on V0 v. [S] plot?

2. why

3. how similar to normal non regulatory micaelis Minton?

4. how do positive and negative allosteric modulators affect the curve?

5. what do modulators change?

6. KEY characteristic of sigmoidal kinetics

1. allosteric enzymes = sigmoidal instead of normal hyperbolic curve

2. usually complex multimer proteins w/ complex reaction

3. still reach saturation at high [S]

4. positive = hyperbolic, shift left (high activity/r state)

5. negative = shifts right (low activity/T state)

6. there are many diff kinds that could change max or rate etc.

7. small changes in modulator concentration can have large affects on activity

aspartate transcarbamoylase (ATCase):

1. what does it do

2. how regulated (4) / how plot move

3. why sigmoidal

4. homo or hetero?

1. catalyses reaction of aspartate

2. has regulatory subunits and catalytic subunits

   1. catalytic bind substrate

   2. regulatory binding change conformation

      1. aspartate and carbamoyl phosphate = positive homotropic (substrate)

      2. ATP = positive heterotropic allosteric modulator → shift left relaxed state / high affinity

      3. CTP = neg heterotropic allosteric modulator → shift right tense state / low affinity

3. binding of substrates aspartate and carbamoyl phosphate = transition from T state →R state

1. ATCase both homo and hetero allosteric kinetic behavior

aspartate transcarbamoylase (ATCase):

1. why does ATP activate and CTP inhibit?

2. what’s the mechanism?

3. what are pathways? what’s usually the final product usually do?

4. rationale behind ATP activating and CTP inhibiting?

1. allosteric control of ATCase by CTP = feedback inhibition

2. CTP is product produced at end of long pathway and acts feedback inhibitor of earlier ATCase reaction to control level of CTP in the cell

3. processes where product of one enzyme becomes substrate of next each step catalyzed by a different enzyme

   1. final product usually heterotropic allosteric inhibitor of earlier enzymes in the pathway

4. rationale

   1. ATP activates = want to make stuff when have energy

   2. CTP feedback inhibitor = don’t want to waste E making too much

1. how covalent modification regulate enzyme function

2. how permanent are covalent mods

1. many different types/affects of cov mods catalyzed by enzymes

2. permanent until removed by another enzyme

1. describe usefulness of phosphorylation in enzymatic regulation and how glycogen phosphorylase is regulated in response to phosphorylation

Phosphorylation:

1. catalyzed by

   1. what do they reconize

2. removed by

   1. specificity

3. how is glycogen phosphorylase regulated by phosphorylation?

1. catalyzed by kinases

   1. specific, kinases recognize seq motifs

2. removed phosphatase

   1. not specific

3. its really complex, but simple version:

   1. glycogen phosphorylase switches b/t active a form and inactive b form by phosphorylating 2 Ser residues

      1. phosphorylase kinase activates = turns b → a

      2. protein phosphatase 1 inhibits = turns a → b

cleavage:

1. how modify enzymes?

2. zymogen

3. chymotrypsin cleavage?

1. inactive precursors can be permanently converted to active enzymes by enzymatic (specific) cleavage

   1. can be autolysis

2. zymogen = proenzyme

3. chymotrypsin and trypsin activated by proteolytic cleavage of their zymogen forms