chapter 10: regulatory strategies

three distinctive and characteristic features of enzymes

high catalytic power, high specificity, highly regulated

factors that regulate enzyme activity

covalent regulation, modulator protein, isozymes, allosteric regulation, proteolytic activation

important enzymes for covalent regulation

kinases and phosphates

serine, threonine, or tyrosine + ATP (with protein kinase) →

phosphorylated protein + ADP

phosphorylated protein + H2O (with protein phosphatase) →

serine, threonine, or tyrosine + orthophosphate

glycogen synthase active form

\-OH

glycogen synthase inactive form

\-P

glycogen phosphorylase active form

\-P

glycogen phosphorylase inactive form

\-OH

enzyme for active pyruvate dehydrogenase + ATP → inactive pyruvate dehydrogenase-P + ADP

pyruvate dehydrogenase kinase

enzyme for inactive pyruvate deydrogenase-P + H2O → active pyruvate dehydrogenase + Pi

pyruvate dehydrogenase phosphatase

phosphorylation is done by

kinases

dephosphorylation is done by

phosphatases

phosphorylation or dephosphorylation alters

protein 3D structures

changes of structures results in

an impact on protein functions

3 examples of covalent regulation

glycogen synthase, glycogen phosphorylase, pyruvate dehydrogenase

PKA

protein kinase A

PKA consists of

two kinds of subunits, 2 large regulatory subunits (R) and 2 smaller catalytic subunits (C)

in the presence of cAMP, protein

dissociates into R2 and 2 active C subunits

PKA activation requires how many cAMP molecules

4

PKA is a

tetramer with 2 different subunits: R2 and C2

which PKA subunit has cAMP binding domain

R subunit (2 cAMP per R)

inactive C is converted to active C through

dissociation of C from R due to the conformational change of R upon cAMP binding

an example of ordered bisubstrate reaction

NADH + pyruvate + H+ (with lactate dehydrogenase) → lactate + NAD+

2 forms of lactate dehydrogenase in humans

H form found in heart, M form found in skeletal muscle

two forms of LDH in humans are

75% identical in amino acid sequences

LDH is a

homotetramer (H4 and M4)

combinations of LDH allows for

different affinities for the substrate or the product

since heart is H4 and H3M, it favors

the conversion of lactate to pyruvate

liver and muscle cells are M4 and M3H, they favor

the conversion of pyruvate to lactate

active muscle tissue becomes anaerobic, produces pyruvate from glucose via glycolysis; needs LDH to

regenerate NAD+ from NADH so glycolysis can continue

lactate produced by active muscle tissue is

released in the blood

M4 works best in the

NAD+ regenerating direction

heart tissue is aerobic and uses lactate as fuel, converting it to pyruvate via LDH and using the pyruvate to

duel the citric acid cycle and obtain energy

examples of LDH tetramers

H4, H3M, H2M2, HM3, M4

in heart, affect of LDH?

H4 factors direction of lactate to pyruvate since heart needs pyruvate for citric acid cycle

in muscle, affect of LDH?

M4 favors NADH to NAD+ since muscle needs NAD+ for continuation of glycolysis

enzymes situation at key steps in metabolic pathways are modulated by

allosteric effectors

allosteric effectors are

usually produced elsewhere in the pathway and they do not structurally resemble the substrate, bind allosteric sites

effectors could be

activators or inhibitors

allosteric enzymes have

oligomeric organization

regulatory effects of effector molecules caused by

structural changes in the enzyme upon binding

feedback inhibition

product E is an allosteric inhibitor of enzyme 1

feedforward inhibition

product B is an allosteric activator of enzyme 4

allosteric proteins can exist in two states

relaxes and tense

R and T states of allosteric proteins exist

in equilibrium

activators favor the direction towards

R state

inhibitors favor the

T state direction

in the allosteric protein model, all subunits of an oligomer must be in

same state

S binds tighter to

R

allosteric regulation example

aspartate transcarbamoylase (ATCase)

ATCase reaction

carboamoyl phosphate + aspartate (with ATCase) → N-carbamoylasparatate + Pi → → → CTP (can be converted to ATP)

ATCase subunits?

has two types, 6 large catalytic (active) subunits and 6 small regulatory (binds CTP or ATP) subunits

ATCaseundergoes a

concerted transition

what does concerted transition mean

all or none conformational change

T state of ATCase

less active, favored by CTP binding

R state of ATCase

more active, favored by substrate binding

what stabilizes the T state of ATCase

binding CTP

R6C6 of ATCase has ___ CTP binding sites per R subunit

one

binding of substrate to ATCase promotes

formation of the active R state

binding of CTP to ATCase

stabilizes the inactive T state

activity of ATCase depends upon

relative concentrations substrates versus CTP

allosteric regulators

shift equilibrium between T and R states

CTP inhibition biological significance

ensures pyriminidine synthesis is turned off when sufficient CTP is available

ATP activation biological significance

ensures that DNA replication occurs only when energy and a high concentration of purines are available

what is the model for allosteric regulation

R and T states

which enzyme is regulated by allosteric regulation

ATCase

proteolytic activation examples

chymotrypsin, trypsin and elastate, blood clotting

chymotrypsin rxn

chymotrypsin (inactive) with trypsin → pi-chymotrypsin (active) with pi-chymotrypsin converts → two dipeptides + A chain of alpha-chymotrypsin + B chain of alpha-chymotrypsin + C chain of alpha-chymotrypsin

many enzymes are active

as soon as they are synthesized and have folded, but some are synthesized in places where they should not be active so they are activated after being transported to the appropriate place

inactive enzyme precursors

zymogens

zymogens are activated by

proteolytic cleavage, which occurs outside the cell

protease cleavage action example

X cleaves A to B, B cleaves C to D, D cleaves E to F, etc

one enzyme can convert 100 substrates to 100 products. if one X is activated, how many F will be produced

100 x 100 x 100

trypsin is synthesized as what where

inactive trypsinogen in the pancreas

trypsin is the

common activator of all pancreatic zymogens

what converts trypsinogen to trypsin

enteropeptidase and trypsin (self-cleavage)

trypsin cleaves

chymotrypsinogen to chymotrypsin, proelastase to elastase, procarbodypeptidase to carboxypeptidase, prolipase to lipase

conversion of zymogen via proteolysis is

irreversible, binding is very strong

conversion of zymogen via proteolysis needs different mechanism to

turn off proteolytic enzymes called specific protease inhibitors

pancreatic trypsin inhibitor binds

to the active site of trypsin, inhibiting the enzyme to prevent its over-reacting

pancreatic trypsin inhibitor is a

substrate analog

pancreatic trypsin inhibitor reaction: lysine 15 binds to

aspartate 189 in trypsin active site, which makes the interaction look like a true substrate

inhibitor of elastase

alpha one- antitrypsin

normal functions of elastase

breaks down elastin to elastic fiber that interacts with collagen to determine mechanical properties of connective tissues

alpha one- antitrypsin binds

irreversibly to elastase and prevents its over-reacting

unrestrained elastase

destroys alveolar walls in the lungs by digesting connective tissue proteins

emphysema is called by

lack of alpha one-antitrypsin and subsequent alveolar walls destruction

people with emphysema have

a hard time breathing

increases the likelihood of someone who is a heterozygote in the defect developing emphysema

smoking

cigarette smoking

oxidized methionine 358 of the inhibitor, which is critical for binding elastase

proteolytic activation

irreversible process and need to control their enzymatic activities through inhibitors

blood clots are formed by a

cascade of zymogen activations

catalytic nature of cascade leads to

large amplification, ensures rapid response to trauma

blood clotting cascade intrinsic pathway

results from rupture of blood vessels

blood clotting cascade extrinsic pathway

results from tissue damage and releasing clotting substances in response to trauma

intrinsic and extrinsic pathways are triggered by different signals, but

two pathways converge at final common pathway

both pathways are

precisely regulated and signals are amplified in both pathways, irreversible

intrinsic pathway of blood clotting

damaged surface causes kininogen kallikren to convert XII to XIIa, XIIa converts XI to XIa, XIa converts IX to IXa, IXa with VIIa converts X to Xa

extrinsic pathway of blood clotting

VII converts VIIa, VIIa with tissue factor produced by trauma converts X to Xa