L9: Enzyme Regulation, Inhibition, Mechanics

continuation of L8 material

reversible inhibitors

competitive, uncompettive or non comp molecules bidning non covalently and can dissocate from enzume

KI IS FOR ACTIVE SITE BININDG, KI’ IS FOR OTHER SITE BINDING

COMPETITIVE INHIBIT

• comp inhib bind to substrate active site forming an EI

• dissociation is on free enzyme ki = [e][i]/[ei]

• can help with blocking protein function, building good drug targets

• more [s] needed for same reaction rate = increases apparent value of km, so half v max is being altered because curve is changing shape

uncomp inhibitors

1. uncomp inhib doesnt bind to substrate binding site, can inhibit ES complex rather than free complex → ESI (enzyme substrate inhibitior compx) ki’

2. reduction of km that is apparent since nothing is converted to product

   1. appears like you are using subtrate more efficiently but since esi complexes cant be broken, youll get a decrease in vmax

   2. no matter how much substrate you throw at the enzyme, it doesnt change the product

   3. vmax and km decrease by same factor

what inhibition is represented by this graph

do comp inhib alter vmax?

they do not due to being overcome by high [s]

• km is apparent instead of proper bc of high level of substrate

• km increase, but vmax is not alterned because you can overcome comp inhibition

mixed inhibition

bind different site from substrate and can bind either enyme or es complex = ESI cannot form product

• for both cases vmax decreases because in both cases youll get some inhiitor binding to an es complex, so there is no product formation from esi complexes

• km depends on which dissociation constant is higher:

  • is es diss is hgiher than free enzyme = mostly comp inhibitior activity = km increases since it stays bound and associates more with free enzyme = more substrate is needed to proceed

  • diss for free enzme is higher than es = mostly uncomp inhibitor = more inhibitor will stay bound to es complex: forming esi complex

pure non-comp inhibitor

type of mixed inhibitor that binds different site from substrate and can bind either enzyme or ES complexes with equal affinity (ki = ki’) = ESI cannot form product

• km does not change because of equal binding of inhibitor to E and ES

• vmax: inhibotr has ability to bind to es complex so it cannot be rescued with high substrate concentration so vmax decreases

want low kd so it stays together

comp inhibit will be best at block any formation of product bc inhibiting es complex will not actually prevent any product happen (needs to bind to es complex) → stopping reaction earlier rather than later

monomeric follow what type of reaction kinetics does it follow

follow simple michealeic kinetics

commited step

the first step in a feedback inhibition pathway where once the reaction occurs, the rest is commited to the conversion of end product

once D is made, you have to continue even though f can change back to e, it cant change back to D, also not e5 bc you need e4 first

feedforward activation

if precuror molecule is accumulationg, they can speed up downstree reactions

why does allosteric enzymes nto follow m-m kinetics

allosteric enzyme velocity to substrate curves differ from typuca curve due to sharp increase of V0 in middle = sigmoidal

• R and T are in equillibim in absence of substrate (T is more stable/common)

• l0 = allosteric constate = T/R ration

homotropic effect

homotropic effect

• substrate and modulator are the same molecule

• value of [s] at which v0 = vmax is referred to as k0.5 instead of km

• can follow concerted or sequential models of cooperativity for enzymes with multiple subunits

• R state hsas rapid increase of activity towards vmax compared to lower activity of t state hence the lag of the sigmoidal curve (black line)

in narrow range of [s] what happens to allosteric enume transition

allosteric enzymes transition from less active to more active state in narrow range of [s]

m-m kinetics vs allosteric, threshold effect means what

allosteric enzymes are more elastic near km than typical m-m enzymes with same vmax

threshold effect = enzyme active increases rapidly once [s] threshod is passed

heterotropic effect

binding of one moleculer (effector) has effect on a different molecule (substrate)

• a psotive effector (atp) would bind R form at regulatory site to stabilite it (more activation), smaller k0.5

• a negative effector (ctp) would bind to T state and stabilize it (less activation), larher 0.5

gout

disease where excess of urate crystallizes in fluid and linig of joints causing painful inflammation

• urate is final product of purine degradation pathway

• mutation in PRS = no feedback inhinition by purine nucleotides = excess purienes

covalent catalysis (dont need mecahnism)

covalent catalysis:

• formation of a transient covalent bond between substrate and enzyme can create unstable intermediate which promotes catalysis

• e.g. nucleophilic (electron rich) group on enzyme attacks an electrophilic group on substrate (dont need to know mechanism)

acid base catalysis

two types of catalysis that involve proton transer either with water (specific) or functional group (general)

• acid catalysis = donation of proton by enzye

• base cata = removal of proton by enzyme

metal ion catalysis, metal activated enzymes, metalloenzymes

metal ions act as cofactors that may promote orientation of bound substrates or sheild stabilize charges.

• can also create a nucleophile by increasing acidity of nearby molecuels like water

• metal activated enzymes = loosely bound enzymes bound to mg2+, ca 2+ or na+, k+)

• metalloenzymes = tightly bound (typicall to zn2+, fe 2+, mn, etc)

covalent catalysis by approximation

reactions iwth two distinct substrates could have enhanced rate b bring them in:

1. close proximinity (no need for random collision)

2. proper orientation (reduces imrpobability of reacting in correct orientation

aka proximity affect

chymotrypsin (know the reaction mechanism), when is it secreted

serine protease enzyme that is secreted by pancrease after a meal, and it selectively cleaves peptide bonds on carboxyl side of trp tyr phe met and ille

• contain ser, asp, and his

• made up of one polypeptide chain that is cleaved into three pieces that are linked by disulfide bonds to form an enzyme with an active site on the surface

how can chymotrypsin activity be measured

measured by cleavage of a substrate to a product that produces a yellow colour

catalytic traid

3 amino acids that create hydrogen bonded network for catalysis (ser on C chain, his on B chain, Asp on B chain)

in active site of chymotrypsin

ser195 in catalytic triad

first step of proposed enzyme reaction mechanism of chymotrypsin

formation of a covalent acyl-enzyme intermediate between ser195 and substrate (promotes cleavage of peptide bond and release of C terminal fragment)

second step of proposed enzyme reaction mechanism of chymotrypsin

enzyme regeneration based on deacylation and release of n-terminal fragment

what is step one of reaction mechanism of chymotrypson based on?

the enzyme binding substrate (step 1) is based on aromatic side chain and specificity pocket alighning

in step 2, where does the proton transfer occur and what does this create?

proton transfer from ser 195 to his 57 = ser195 is now a nucleophile that attacks carbonyl carbon on polypeptide backbones

what is formed after steps 1 and 2 reaction mech of chymotrypsin

formation of covalent transient tetrahedral intermedia that resembles transition state conformation

• C-O bond (is longer than c=o) allows negative oxygen to interact with oxyanion hole in active site

  • forms hydrogen bonds with nh groups of ser 195 and gyl 193

what is step 3 of chymotrypson mechanism

imiidole ring of his57 → acid catalys → donates proton to N of peptide bond → cleaves - > c terminal is released

• n terminal frag remains bound as covalent acyl-enzyme intermediate

step 4 of cymotrypsin

water donates proton to his → free oh- → attacks carbonyl carbon on acyl-enzyme = forms second tetra intermediate thats stabilitized by oxyanion hole

step 5 of chymotrypsin adn step 6

protonated his 57 donates h → cleaves covalent bond of ayl enzyme intermediate, product 2 (n terminal gragment is released

step 6: catalytic triad is regenerated

serine proteases have ___ substrates. define two of them

serine proteases have diff substrates hence substrate specificity

typrsin = cleaves at peptide bonds neighbouring postively charged amino acids

elastase = specific for elastin (rich in glycine and alanine)

binding pocket has huge affect on what they bind to

enolase

metalloenzyme that is part of glycolytic pathway, catalyzes dehydration of 2 phosphoglycerate to phopyruvate

• two monoers, each have active site that contain two divalent metal ions

enolaste catalytic strategy: step 1

step 1:

lys345 acts as base to remove proton at C2 of substrate → enolate intermediate

enolaste catalytic strategy: step 2

glu211 acts as general acid, donates H to oh leaving group → -h20 → forms phosphoenolpyruvate

coenzyme dependent redox reactions

transfer of e between coenzyme and substrate so one is ox and other gets red

• responsble for muc of energy converion in cells like metabolic pathways

• fadh and nadh reactions

metabolite transofmration reactions

chemical transf of metabolites to reactiove intermediates

• needed for anabolic and catabolic pathways like isomeration, condensation, hydrolysis oor dehydration

reversible covalent mod reacitons

atta or removal of molecular tags that control activity

• cell signalling or gene expression, phosphorylation or methylation

phosphorylation

occurs on ser, thr and try or his sometimes

• added by kinase removed by phsophatase

• activate or inactivate enzymes

c bc ser can only be phsophorylated and so if it cant be, then it is inactive

why are enzymes synthesized in inactive form? proteolysis

to prevent non specific digestion of cellular protein

• synthesized as inactive forms (zymogens) that can be irreversibly activated by cleavage

active form of pepsinogen is caused by: and what is autocleaveage stimulated by

celeaving the 44 aa n terminal fragment

autocleavage is stimulated by low stomach pH

chrymotrypsin is first procesed by what?

trans cleavage by trypsin formting partially active enzyme, leading to autcleavage that fome active enzyme

adenylytaion vs deadenlyation

addition of nucleoside monophospate group vs removal of it (like amp)

uridylylation

attached of ump to regulate adenylation or deadylation