Lesson 13: Covalent Modification, Proteolysis, & Allostery

covalent modification

rapid, reversible change to 1 or more residues by forming/breaking a covalent bond

most common form of covalent modification

phosphorylation

phosphorylation (+ chemical formula of added group)

addition of a phosphate group from ATP or GTP; [PO4]2-; draw a phosphate!!

phosphorylation catalyzed by

kinases

kinases

catalyze phosphorylation by hydrolyzing ATP to remove γ-phosphate

γ-phosphate

terminal phosphate of ATP

dephosphorylation

removal of phosphate

dephosphorylation catalyzed by

phosphatases

phosphatases

hydrolyze phosphate-ester bond

structural change from phosphorylation

adds negative charge -> changes electrostatic interactions & conformation

timing advantage of phosphorylation

addition / removal catalyzed by 2 different enzymes -> rate can be tightly regulated

energetics of phosphorylation

-50kJ/mol per phosphate, half is conserved in phosphorylated protein

sensing function of phosphorylation

enzyme regulation linked to cell's energetic state (ATP availability)

how does phosphorylation amplify signals

1 kinase can phosphorylate hundreds of proteins

proteolytic cleavage

activate an enzyme by cleaving part off of it

proenzyme

inactive enzyme before cleavage

zymogen

proenzyme of a protease

proteolytic cleavage to form pepsin (steps)

pepsinogen has a segment blocking active site -> cleaved into active pepsin

chymotrypsin proteolytic cleavage mechanism

chymotrypsinogen (preproenzyme) cleaved by trypsin -> π-chymotrypsin (proenzyme), self-cleaves -> 𝛼-chymotrypsin

allosteric enzymes

activity modulated by cell signals / metabolic activity

feedback inhibition

product from later in a metabolic pathway inhibits an enzyme early in the pathway

allosteric enzyme structure

multiple subunits (catalytic and regulatory), multiple active sites, allosteric sites

effector

modulator of allosteric enzyme

catalytic subunit

subunit that catalyzes reaction

regulatory subunit

subunit that receives signal + modulates activity; contains allosteric site

allosteric site

binding site for modulators

Michaelis-Menten for allosteric enzymes

do not follow Michaelis-Menten

shape of saturation curve for allosteric enzymes & why

sigmoidal due to cooperativity / inter-subunit interactions

states of allosteric enzymes

T & R states; R state favors substrate binding and is more active

ATCase

aspartate transcarbamylase

ATCase pathway

ATCas catalyzes carbamoyl phosphate + L-aspartate -> Pi + N-carbamoyl-L-aspartate, the first step in the pyrimidine pathway

ATCase inhibited by

CTP

committed step

reaction early in a pathway after which you can't go back; commits pathway to completion

ATCase structure

12 subunits: 3 regulatory dimers, 2 catalytic trimers

T & R states of ATCase

catalytic trimers are further apart in R state, closer together in T state

ATCase activated by

ATP

glycogen phosphorylase

removes glucose from the ends of glycogen using phosphate, forms glucose-1-phosphate

glycogen phosphorylase structure

homodimer (2 identical subunits); active site buried in T state, accessible in R state

covalent modification of glycogen phosphorylase

phosphorylation of identical Ser residues pulls homodimers from each other -> R state

allosteric modulation of muscle glycogen phosphorylase

activated by AMP, even if dephosphorylated; inhibited by ATP and glucose-6-phosphate

allosteric modulation of liver glycogen phosphorylase

inactivated by glucose, no allosteric activators

acetylation (+ chemical formula of added group)

addition of acetyl group; ---C=OCH3

adenylation

addition of adenosine from ATP

methylation (+ chemical formula of added group)

addition of methyl group; ---CH3

myristylation

addition of myristic acid fatty acid chain

ubiquitination

addition of regulatory protein ubiquitin

uridylation

addition of uridine from UTP