Biochem CH 7: the behavior of proteins: enzymes, mechanisms, and control

feedback inhibition

• process by which the first product of a series of reactions inhibits the first reaction in the series

• prevents excess of final product

• prevent accumulation of intermediates in the pathway

• Ex) Asparte transcarbamoylase(ATCase) in catalyzing CTP

Carbomoyl phosphate + Aspartate--(ATCase +HPO4²- leaves)—>carbomoyl aspartate—> series of reactions—> nucleotide with CTP—> excess CTP inhibits first step of reaction

Cytidine triphosphate (CTP) reaction

• ATP acts as the activator binds to the same regulatory site at CTP

• ATP activates ATCase when CTP is low, making the enzyme work more efficiently and reducing cooperativity so the cell can produce more CTP.

• reaction rate increases at lower aspartate concentrations

• curve becomes more hyperbolic

explain the substrate/velocity curve for aspartate and ATCase when an activator is present

normal sigmoidal curve

explain the substrate/velocity curve for aspartate and ATCase when no A or I is present

• Excess CTP inhibits reaction

• curve shifts to right= higher concentration of Aspartate needed to reach the same reaction rate without an inhibitor

• ultimately the Vmax is the same as the reaction without an I just takes more substrate to reach that

explain the substrate/velocity curve for aspartate and ATCase when I is present

K-systems

• combinations of allosteric enzymes and inhibitors or activators, in which the presence of the inhibitor/activator changes the substrate concentration that yields Vmax

• Vmax stays the same in both cases while K0.5 differs

lowers, less, raise, more

In K-systems activators ____ K0.5 meaning ___ substrate needed to reach K0.5 while inhibitors ____ K0.5 meaning ___ substrate needed to reach K0.5

V-systems

• combinations of allosteric enzymes and inhibitors or activators in which the presence of the inhibitor/activator changes the maximal velocity of the enzyme but not the substrate level that yields one-half Vmax

• K0.5 stays the same while Vmax differs

increase, more, decrease, less

In V-systems activators ____ Vmax meaning ___ enzymes active (R-state)to catalyze. Inhibitors ____ Vmax meaning ___ enzymes active to catalyze

quant, behavior

binding of substrates, inhibitors, and activators, change ___ structure of allosteric protein and thus ____

allosteric effector

a substance- substrate, activator, or inhibitor- that binds to an allosteric enzyme and affects its activity

homotropic effects

allosteric effects that occur when several identical molecules are bound to a protein

Ex) ATCase with aspartate

heterotopic effects

allosteric effects that occur when different substances are bound to a protein

Ex) ATCase wih ATP and CTP (activator/inhibitor)

concerted model

a description of allosteric activity in which the conformations of all subunits change simultaneously

R-state

relaxed confirmation, binds substrate tightly

T-state

tight/taut confirmation, binds substrate tightly

T/R, L is usually bigger meaning more enzymes exist in the T-state

what is L?

K

Dissociation constant of enzyme and substrate, enzymes affinity for substrate, how tightly substrate binds to enzyme

Kr<Kt

affinity for substrate is higher in R form that T form

Kr/Kt

what is c

Kr/Kt=c=0 meaning T’s affinity for the substrate is infinitely small so substrate will not bind at all

what happens when Kt= infinity

• R-state higher affinity = holds onto substrate more tightly that T-state

• as substrate binds R-state free form is removed

• production of more R-form from T-form to replace what is “lost”

• more R means more substrate binding is possible

explain the equilibrium state between R and T when a small amount of R-state is present

L, sigmoidal

Monod-Wyam_changeux model: As ___ increases, free form T is more highly favored the shape becomes more ___ (increase in T)

c, sigmoidal

Monod-Wyam_changeux model: as ___ decreases, higher affinity between substrate and R form, the shape becomes more ___ (increase in Kt)

• activator binding R form and stabilize

• equilibrium shifted in favor of R form

• enzyme in active form mean less need for S to shift equilibrium in favor of R form

• S binds easily b/c more binding sites

• less cooperativity in the binding of S is seen= hyperbolic substrate curve

explain the Effects of binding activators with the concerted model graph

cooperative binding

the first substrate binding event makes the next one easier.

normal cooperativity S must shift equilibrium from T—>R= sigmoidal substrate curve

explain the Effects of no A or I with the concerted model graph

• inhibitor stabilizes T form

• T form is favored

• more substrate needed to flip enzymes to R-state

• curve is sigmoidal and shifted right b/c K0.5 increases and cooperativity increases

explain the Effects of an Inhibitor with the concerted model graph

K0.5

the substrate level at one-half Vmax in a K system

sequential model

• description of the action of allosteric proteins in which a conformational change in one subunit is passed along to the other subunits

• Ex) substrate binding— T—>R one subunit—→ other subunits turns to R

negative cooperativity

• cooperative effect whereby binding of the first ligan to an enzyme or protein causes the affinity for the next ligand to be lower (less likely to bind next ligand)

• Ex) Tyrosyl tRNA: binding of ligand to first unit inhibits binding of a second molecule to the other subunit

phosphorylation

phosphate group added to some substance

• in enzymes can increase or decrease activity

allosteric sites

binding sites on a target molecule for allosteric effectors

protein kinases

class of enzymes hat modify a protein by attaching a phosphate group to it

kinase

class of enzyme that catalyze a transfer of a phosphate group to some substrate

zymogens

an inactive protein that can be activated by a specific hydrolysis of peptide bonds and become permanently (irreversible)

• enteropeptidase activates trypsinogen to trypsin

• trypsin activated chymotrypsin (cleave between Chym Arg 15 and Ile 16)

• cleavage produces pie-chymotrypsin= fully active and two separate chains held together by 1 out of 2 S-S bonds

• pie-chymotrypsin cleaves further by removing 2 dipeptides (ser14-Arg15 and Thr147 and Asn148)

• final product = a-chymotrypsin, has 3 polypeptide chains held together by 2 S-S bonds

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explain how trypsinogen and chymotrypsin are inactive in pancreas and become active in small intestine

caspases

• family of homodimer cysteine proteases responsible for many processes including cell death (apoptosis)

• first produced in inactive form, procaspases

• which amino acids are present on enzyme active site

• what is spatial relationship of these AAs

• mechanism by which AA residues catalyze the reaction

what determines how the active site increase the enzyme rate of reaction

model systems

provides the essential features of a reaction in a simple form that is easier to work with than one found in nature

the hydrolysis of peptide bonds adjacent to aromatic amino acid residues in the protein being hydrolyzed; other residues are attacked at a lower frequency and hydrolysis of esters in model studies in the laboratory b/c the amide and ester bond are similar enough

what does chymotrypsin actually catalyze

step 1= burst of of p-nitrphenyl/ lag of acetate

step 2= steady formation of acetate

chymotrypsin catalysis of p-nitrophenyl

serine protease

class of proteolytic enzymes which a serine hydroxyl plays an essential role in catalysis

Ex) Chymotrypsin needs the serine exposed

• DIPF forms a covalent bond with Ser195 R-groups, without available Ser195 it cannot function as a serine protease

How is chymotrypsin inactivated by DIPF

labeling

covalent modification of a specific residue on an enzyme

DIPF (Seri195) and TPCK(His57)

What inactivates chymotrypsin

TPCK forms a covalent bond with His57

How does His57 inactivate Chymotrypsin

nucleophile

donates electrons

electrophile

accepts electrons

• Ser195 acts as a nucleophile His57 abstracts a proton from Ser95 OH group

• Ser195 attacks carbonyl cation of peptide group

Mechanism of catalysis for Chymotrypsin Step 1

Carbonyl oxygen becomes an oxyanion, tetrahedral intermediate forms

Mechanism of catalysis for Chymotrypsin Step 2

tetrahedral intermediate collapses

• His57 donates proton to leaving amino group

• C-N bond breaks forming acyl-enzyme intermediate

Mechanism of catalysis for Chymotrypsin Step 3

water acts as new nucleophile H-bonds to His57

Mechanism of catalysis for Chymotrypsin Step 4

water attracts acyl carbon another tetrahedral intermediate forms

Mechanism of catalysis for Chymotrypsin Step 5

• bond between Ser195 and carbonyl carbon breaks

• product with carboxyl groups released and Ser195 and His57 return to original H-bonded state

Mechanism of catalysis for Chymotrypsin Step 6

nucleophilic substitution reaction

reaction in which one functional group is replaced by another as a result of a nucleophilic attack

Sn1 nucleophilic attack

• unimolecular nucleophilic substitution reaction, rate of reaction follows 1st order kinetics

  • slow part= breaking between R and X

  • fast part= addition of nucleophile Z

• only R-X matters for rate b/c R-X has to fall apart before the nucleophile can be added. So raising Z won’t matter because R-X has to break apart first

• can lose stereospecificity(only 1 3D shape) because the nucleophile can attack from either side once the bond breaks

Sn2 nucleophilic attack

• bimolecular nucleophilic substitution, rate of reaction follows second order kinetics

• both R-X and Z matter for the rate

  • more R-X= more molecules available to be attacked

  • more Z= more nucleophiles attacks happen faster

• Keeps stereospecificity b/c nucleophile must attack from opp side of the leaving group forcing a single orientation

general acid-base-catalysis

• a form of catalysis that depends on transfer of protons (Bronsted Lowry definition)

  • acid=proton donor

  • base=proton acceptor

general acid catalysis

amino acid donates H to substrate

general base catalysis

amino acid takes H from substrate

metal-ion catalysis

form of catalysis that depends on the Lewis definition of an acid as an electron pair acceptor and base as an electron pair donor

• catalyzes hydrolysis of C-terminal peptide bonds of proteins

• Zn polarizes the carbonyl group making it susceptible to attack by water and allowing hydrolysis to proceed more rapidly

how does Zn II work with metal ion catalysis

absolute specificity

catalyze reaction of only one substrate to a particular product (lock and key)

relative specificity

catalyzes reaction of structurally related substrates to related products more flexibility in active sites (induced fit model)

sterospecific enzymes

binding site shapes of stereospecific substrate and not its mirror image

transition state

intermediate structure between the substrate and the product

transition state analogues

• synthesized compounds that mimic the form of the transition state of an enzyme reaction

• Ex) L-proline (tetrahedral carbonyl)—> transition state p(planar carbonyl)—D-proline(tetrahedral carbony but H and COO- reverse) via proline racemase

coenzymes

nonprotein substance that take part in enzymatic reactions and are regenerated at the end of the reaction

cofactors

nonprotein helpers that participate in enzyme reactions and are regenerated afterwards

• coenzyme

• metal ions