Biochem Exam 3

enzyme

• specific 3d and quaternary structure

• not consumed during rxn and can be reused

• some may require cofactors or coenzymes to function

• change rxn rate but NOT deltaG standard or rxn equilibrium

  • lowers activation energy deltaG++

oxidoreductase

• transfer of e (hydride ions or H atoms)

transferases

• group transfer rxns

hydrolases

• transfer of functional groups to water

lyases

• addition of groups to double bonds

• formation of double bonds by removal of groups

isomerases

• transfer of groups within molecules to yield isomeric forms

ligases

• formation of

  • C-C

  • C-S

  • C-O

  • C-N

• by condensation rxns coupled to ATP cleavage

stabilization

• TS (EX++) must be stabilized

• ES must be destabilized/less stable than TS

  • entropy loss raises energy of ES

  • destabilization of ES thru strain/distortion

  • desolvation to raise energy

• makes gap btwn ES and TS smaller (lowering activation energy)

catalytic power

• ratio of catalyzed rate/uncatalyzed rate

enzyme activity

• catalytic power

• sensitivity (pH, temp)

• specificity

  • bond

  • group

  • geometrical

  • stereo

  • co-factor

• regulation

cofactor

• non-ptn chemical components required for enzyme activity

coenzyme

• organic, non-ptn molecule

• assist enzymes by carrying chemical groups or e

• derived from vitamins

• X ARE COFACTORS BUT NOT ALL COFACTORS ARE X

• ex: NAD+ derived from Vitamin B3

co-substrate

• specific subtype of organic cofactor (coenzyme)

• binds loosely and transiently to enzyme

• dissociates and changes chemically during rxn cycle

• ex: NAD+ reduced to NADH

apoenzyme

• ptn portion of an enzyme

• typically inactive

• no cofactor bound

holoenzyme

• complete catalytically active enzyme

• cofactor bound

Vmax

• max rate of rxn when enzyme saturated w substrate

• theoretical limit—never reached

• no more enzymes available; most present as ES complex

Km

• michaelis constant

• [S] at which rxn velocity is half of Vmax

• reflects affinity of enzyme for its substrate

• implies half of total active sites are filled with substrate

• small means high affinity even at low [S]

• independent of enzyme concentration

lineweaver burk

• double reciprocal

• 1/Vmax y-int

• -1/Km x-int

Kcat

• turnover freq

• number of conversions of S to P over time

• allows for comparison of rate limiting steps

catalytic efficiency

• Kcat/Km

• measures how effectively an enzyme converts substrate to product

• combines speed w binding affinity

• HIGHER RATIO = MORE EFFICIENT

enzyme activity

• moles of substrate converted over time

• measure of quantity of active enzyme present under defined conditions

specific activity

• activity of enzyme per milligram of total ptn

• measurement of purity of enzyme

• total enzyme activity (U) / Total ptn (mg)

• HIGHER X, MORE PURE; doesn’t dictate that it is completely pure

reversible enzyme inhibition

• competitive

• noncompetitive

• uncompetitive

irreversible inhibition

• when inhibitor binds to enzyme permanently disrupting activity

• often by forming covalent bond

competitive inhibition

• only one where increased [S] can restore activity towards Vmax

• inhibitor competes w substrate for active site

• Vmax unchanged

• Km increased (less affinity for S)

• intersect at y axis for lineweaver

noncompetitive

• allosteric; binds to other site

• alters shape and reduces activity

• Vmax decreased

• Km unchanged

• intersect at x axis for lineweaver

uncompetitive

• binds to ES complex but not to free enzyme

• Vmax and Km decreased (higher affinity)

• don’t intersect for lineweaver

allosteric regulation

• effector binds at allosteric site to promote/inhibit enzyme activity

• sigmoid-shaped kinetics (S-shape)

• x enzymes are usually oligomeric

covalent modification

• addition or removal of chemical groups

• many are reversible

• ex: phosphorylation/dephos, acetylation, adenylation, methylation/demeth

transcriptional control

• gene expression can be upregulated or downregulated in response to cellular needs

• ex: lac operon in prokaryotes

isoenzymes

• different molecular forms of same enzyme that catalyze the same rxn but vary in aa sequence

• allow precise regulation of enzyme activity

zymogen activation

• inactive precursor forms of enzymes that require biochem activation by proteolytic cleavage

• irreversible

• ex: chymotrypsinogen—converted into active chymotrypsin in small intestine by cleavage of Arg where it helps ptns digest

compartmentation

• enzymes, substrates, regulatory molecules physically separated or located close together

• ex: accumulation of ATP on one side of endothelial cell causes actin polymerization which leads to migration of the cell

cooperativity

• interaction btwn different subunits of enzyme or ptn in response to ligand binding

• affects the binding affinity of other subunits

positive cooperativity

• amplifies enzyme activity by increasing affinity of S to E once first molecule binds

• makes it easier for additional molecules to bind

• sigmoidal curve

negative cooperativity

• lowers enzyme activity by reducing affinity of S to enzyme

• decreases likelihood of further binding after first molecule attaches

• hyperbolic curve

allosteric activator

• shift kinetic curve to left

• stabilizes high-affinity R state so same velocity is reached at lower substrate conc

allosteric inhibitor

• shifts kinetic curve to the right

• reflects decreased apparent affinity as x stabilizes the T-state

MWC model

• ptn already exists in equilibrium btwn T and R states before ligand binds

• all subunits switch together—ptn either all T or all R

• S binds more strongly to 1, usually R

  • shifts equilbrium toward that state

• ONLY POSITIVE COOPERATIVITY

• ex: hemoglobin

  • O2 substrate stabilizes high-affinity R producing positive cooperativity

KNF model

• ligand binding induces conformational change in 1 subunit

• change transmitted to neighboring subunits one at a time

• different subunits can temporarily have diff conformations

• change may inc or dec affinity of neighboring

• can have positive or negative cooperativity

covalent catalysis

• enzyme forms temp covalent bond w S

• creates more reactive intermediate that facilitates the rxn

• ex:

  • chymotrypsin cleaves carboxyl side of large hydrophobic or aromatic aa; Phe, Met, Tyr, and Trp

  • reversible phosphorylation of Ser, Thr, and Tyr residues

low barrier hydrogen bonds

• special type of H bond where proton is more equally shared btwn donor and acceptor atoms rather than strongly associated w 1

metal ion catalysis

• metal ions in enzyme’s cofactors interact w S

• stabilize TS and orient S for rxn

• ex: thermolysin

acid base catalysis

• proton is transferred to catalyze rxns

• histidine often used; pka near 7

• ex: serine and aspartic acid proteases

chymotrypsin

• hydrolase class

• uses covalent and acid base catalysis

  • forms temp covalent w S

  • uses serine as Nuc to attack S’s carbonyl carbon

glycogen phosphorylase

• contains active and allosteric sites

• regulated by phosphorylation on Ser14 of each subunit

• cleaves glucose units from nonreducing ends of glycogen through phosphorylsis rxn to convert into cellular fuel

• allosteric regulation and covalent modification

  • inhibited by ATP and glucose-6-P

  • activated by AMP

• activated—R state

• inactivated—T state, reduced access to active site

GP mechanism

1. acid catalysis via Pyridoxal 5’-phosphate on GP

2. Oxocarbenium intermediate

3. nucleophilic attack, releasing alphaD-glucose-1-phosphate

adenylyl cyclase

• converts ATP to cAMP and pyrophosphate

• rxn driven forward by hydrolysis of pyrophosphate (PPi)

• activated by G-protein subunit signal binding

• cAMP binds to and activates protein kinase A, secondary messenger

hemoglobin

• tetrameric oxygen-transporting ptn—binds 4 oxygen molecules

• sigmoidal, cooperative oxygen binding curve

  • binding of oxygen to first subunit makes binding to other subunits more favorable

• n_H > 1 represents positive cooperativity

• O2 binding shifts x from T to R

  • salt bridges stabilize T but break during transition to R

• R binds additional O2 more easily

  • positive cooperativity

  • sigmoidal curve

• change in O2 saturation is efficient even w small drop of pO2

negative allosteric effector

• 2,3-BPG

• binds at site distant from Fe where oxygen binds

• reduces affinity of Hb for oxygen

• stabilizes T state

bohr effect

• O2 binding antagonized by H+ and CO2 in the tissues

• lower pH and increased CO2 stabilize T state

• curves shifted to right

fetal hemoglobin

• fetus depends on mother for oxygen

• gas exchange occurs in placenta

• contains gamma chains instead of Beta

  • alpha2gamma2 structure

  • higher affinity to oxygen than regular hemoglobin

• serine instead of histidine at position 143

• lacks 2 (+) charges and binds 2,3-BPG less tightly

• looks like myoglobin in O2 binding behavior

sickle cell anemia

• Glu6Val mutation in Beta-globin (HbS)

• causes deoxy-HbS polymerization

• distorts RBC into crescent shapes under low-oxygen/acidic conditions

  • more rigid than regular RB

• rigidity and aggregation lead to:

  • blockage of capillaries

  • circulation impairment

  • tissue damage

  • premature RBC death leads to shortage/anemia

myoglobin

• oxygen-storing metalloprotein

• monomeric

• one prosthetic heme group which binds to single oxygen

• demonstrates hyperbolic oxygen binding curve

  • non-cooperative binding

• higher binding affinity to oxygen compared to hemoglobin

comparison of hemoglobin and myoglobin

• O2 binding changes the conformation

• pulls Fe2+ into heme plane, moving proximal histidine and its attached helix towards heme plane

• Myoglobin

  • change remains local bc monomeric

• Hemoglobin

  • movement transmitted across subunit interfaces since tetrameric

  • disrupting T-state salt bridges and promoting T → R transition