Lecture 6: Enzyme Mechanisms and Kinetics

example of an enzyme mechanism

chymotrypsin

• example of “serine protease” → enzyme protein

• hydrolysis cleaves polypeptides on C-terminal side of aromatic amino acids

• mechanims → both acid/base catalysis and covalent intermediate

describe the chymotrypsin mechanims: free enzyme

• asp acts as strong base → O tugs NH on his (interaction makes his a stronger base) → which tugs H on ser (making ser a stronger acid) → making it reactive

• change relay system

describe the covalent transition state

• bond cleaves

• H on ser has been transformed

• forms covalent bonds with substrate

what is the specificity of Serine proteases determined by

binding pocket

discuss enzyme kinetics

• allows one to predict rates, given [S]

  • as [S] increase so does the rate

• reaction mechanisms

• reveals regulation

• as initial stages, [S] >> [P] → rate is ~constant

• velocity (delta P/ delta t) is dependent on [S] at low [S]; subtsrates to Vmax at high [S]

describe reaction velicity vs [S] what is the relationship between v_initial and [S]

Michaelis-Menten rate law

think about the conceptual view of enzyme saturation

what does the michaelis mentain equation describe

observed behavior

what is the meaning of Km

• always [S] at which velocity is half maximal

• often a measure of affinity of E for S

• if k₂<< k_-1 then:

  • Km = (k_-1+k₂)/k₁ = (k_-1)/k₁ = Ks

• where Ks is the dissociation constant for ES → E+S

  • low Km = high affinity

what is the meaning of Vmax

• always maximum velocity when E is saturated with S (depends on amount of E)

• often measure of rate constant for catalytic step

• when k₂<k₁ then

  • Vmax = k₂[E_total]

• for complex reactions k_cat = rate constant for overall reaction

• k_cat = turnover number = # molecules S → P per second per E molecule

  • how fast an enzyme can complete one round of rxn

describe enzyme activation by proteolysis (protein breakdown)

• examples

  • digestive enzymes

  • blood clotting factors

  • programmed cell death

  • peptide hormones

• proteolytic activation is irreversible

example: proteolytic activation of chymotrypsin

• cleavage results in movement of amino acid that “plugs” active site away from active site

describe regulation of proteases by inhibitor peptides

small peptides that bind to the active site and inhibit protease function

• example

  • anti-elastase, produced by the lungs, blocks connective tissue breakdown by elastase (emphysema)

describe the activation cascade in blood clotting

• clotting factors (except fibrin) are serine proteases

• activated by proteolytic cleavage

• enormous amplification of very small signal

describe regulation of enzymes by covalent modification

• usually reversible

• can activate or inhibit enzyme

• types

  • phosphorylation

  • adenylation/uridylylation

  • ADP ribosylation

  • methylation

  • acetylation

describe regulation of glycoge phosphorylase by phosporylation

• glycogen phosporylase cleaves glycogen

  • → releases glucose for energy

• reegulated by phosphorylation

• also regulated by hormone triggered phosphorylation cascafe and allosteric regulation

enzyme inhibitors (2 types)

• non-specific: urea, detergents etc

• specific: interact with one enzyme

  • antibodies (inibits enzyme only in bacteria)

  • drugs (lovistatin inhibits HMG-CoA redutase → insecta)

  • herbicides (target ezymes unique to plants or insects)

  • pesticides ^^

  • metabolites (feedback regulation)

• specific inhibitors often resemble substrate or transition state

• can be reversible or irreversible

discuss reviersible enzyme inhibition

inhibitor - a compound that binds E and interferes with its activity

• can act by preventing formation of the ES complex or by blocking the chemical reaction that leads to the formation of product

what are the types of inhibitors

competetive: binds only to free E not bound to S (most common

non-competetive: can bind E or ES; not substrate analogs and do not bind same site as S

Determining Km and Vmax: Line weaver-Burk Linear plot

• x intercept = -1/Km

• y intercept = 1/Vmax

• reciprocal of Michaelis Menten rate law

  • 

Describe competetive inhibitors

• inhibitor binds to active site (resembles S)

• Alters Km but not Vmac

  • competition appears to decrease affinity

• ex. used to treat poisonings

Describe non-competitive inhibitors

• alters Vmax but not km

  • cant drive to same vmac even with excess S

• ex. histidine biosynthesis

• 

describe allosteric regulation

• modulators (activators or inhibitors) bind to allosteric site of enzyme ( seperate from actove site)

• binding of modulator alters enzyme activity

• can affect either Km or Vmax

• allostery generally results from interactions among subunits of multimeric proteins → quaternary structure

describe cooperative allosteric regulation

• multimeric proteins

• binding of effectir (modulator) to one subunit alters activity of others

• ex. homotrophic allostery: effector = S

  • heterotrophic: effector and S are different

discuss cooperativity and kinetics

coopertivity does not follow michaels-menten kinetics

• still can experimentally determine Km and Vmax

Discuss Allosteric regulation by heterotrophic (non-S) effectors (modulators)

• ATP: activates (energy available)

• CTP: inhibits (dont need rxn when enough CTP is present)

what is feedback inhibition

what is cumulatove feedback inhibition

• cumulative effect of allosteric regulators decreases G.S. activity in stepwide fashion

what are irreversible inhibitors

• destroys enzyme

• “suicide substrate”

• ex. penicillin → inactivates enx of cell wall synthesis