bchm exam 2

6 bonds of hemoglobin (Hb) and when they occur

all covalent

4 bonds between the Fe2+ and the nitrogen atoms in the 4 pyrrole rings of the porphyrin ring

1 proximal histidine bond with His F8

1 bond to oxygen, which bonds with distal histidine His E7

how do we know myoglobin binds more tightly to o2 than Hb?

Kd = P50 = [L] at Y = 0.5 : Kd is equal to the ligand concentration [L] when exactly half of the binding sites are bound by ligand (Y = 0.5).

lower Kd means less substrate needed to occupy the same number of sites (50%)

what is the shape of Mb vs Hb curve? draw both. why is Hb shape that way?

mb = hyperbolic

hb = sigmoidal - favors release of O2 at low conceentations (left side of graph) and binding at high concentrations (rigth side of graph). this shape allows for change in binding affinity.

Kd and Ka: what does each represent (eqn), relationship, and units

Ka: association constant, for P + L → PL. Ka = [PL] / [P][L]. units: 1/M

Kd: dissoc constant, for PL → P + L. Kd = [P][L] / [PL]. units: M

Kd = 1/Ka

strong binding and weak binding in terms of Kd

strong: Kd < 10 nM

weak: Kd > 10 uM

theta or Y = ?

[L] / Kd + [L]

Hb and Mb have similar ______ because of similar _____ but not similar ______ (only _____ identical).this is because there are: (define each)

• invariant residues

• conservative substitution

simialr teritary structure due to similar function, but not similar primary structure (only 18% identical).

• invariant residues: exactly the same between the two

• conservative subsitution: substitued with a similar amino acid (ex. asp and glu are both acidic)

what three conformational chanegs happen when O2 binds and Hb swtiches from T to R state

• 15 degree rotation of a1b1 subunit from a2b2 subunit

• central cavity becomes smaller

• interfacial subunit interactions (salt bridges) are broken

where does O2 bind in Hb

at the 4 heme groups

what residues are involved in interfacial interactions

His146 (β), Asp94 (β), Lys40 (α), and Arg141 (α)

does lower pH favor r or t state? explain mechanism

lower pH → more H+ → residues get protonated (ex. His146 is positive) → neutral/positive AAs become more positive and can form salt bridge with Asp94 on the same B subunit → t state favored

what is the bohr effect

the response to an increase in CO2 or lower pH: favors the t state and lowers the binding affinity of O2, favoring release of O2

does changing His146 to Glu favor t or r state? explain

Glu = - → unable to form salt bridge with Asp94 → r state favored

what makes the t state stable? what makes the r state stable? what makes the r state more stable than t

t state: interfacial subunit interactiosn

r state: fe-o2 bonds

fe-o2 bonds with 4 ligands (o2) bound is more stable than t state

carbonic anhydrase rxn, why it happens, and its effect on Hb

CO2 + H2O → H+ + HCO3- . turns CO2 in blood (which owuld cause bubbles) into H+.

more H+ / lower pH → Hb protonated → t stare favored → lower bidng affinity

homotropic vs heterotropic effector

positive vs negative effector and how each shifts the curve and what state is favored

homo - binds to same site as ligand (active site)

hetero - binds to an allosteric/different site

positive - increases enzyme activity, leftward shift (decreases P50), T state favored

negative - decreases enzyme acitivity, rightward shift, R state favored

what kind of effector and where does it bind?

Oxygen:

H+

CO2

2,3-BPG

CO

O2 = ligand = positive homotropic allosteric eff

H+ = negative hetertropic allosteric eff - binds to basic amino acids in subunit interface and casues them to be more pos → stronger interaction w their acidic AA pair

CO2 = neg hetero. reacts with uncharged R-NH2 groups and forms NH-COO- which is charged, created more interfacial subunit interactions/salt bridges and favoring t state

BPG = neg hetero. binds in cavity, keeping it in t-state and lowering O2 affinity. when O2 binds, it forces r-state and expels BPG.

CO = neg homo

write Hb and H+ reaction and explain what happens if pH increases.

Hb4O2 + nH+ → ← HbnH+ + 4O2

if pH increases, H+ decreases, rxn shifts left, R state favored + binding affinity increased

what three amino acids does BPG bind to and what makes fetal Hb different

H143, Lys82, His2

fetal Hb has Ser143 instead of His143, so BPG binding affinity is decresed. its called a2g2 instead of a2b2

how can u seen pos cooperativtiy in a binding curve

sigmoidal

hill coefficinet n possibile numbersand what they mean

n = 1 → no cooperativty

n > 1 = pos cooperativity, ie binding of one ligand increases bindnig affinity at other sites

n < 1 → negative cooperativity

hill plot x and y axis

logpO2 and log (Y/ 1 - Y), Y or theta is the fractional saturation

concerted model vs sequential model: what do they model, how are they diff, and what is the limitation of each

models for cooperativity in o2 binding

concerted model: Hb goes back and forth (in equilibrium) between t state and r state, with r state favored as more O2 binds

• limitate: t state w o2 bound doesnt acc exist

sequential: when o2 binds, the subunit gets coverted to t state

• limitation: Hb with some subunits in r state and some in t state (ie an inbetween r and t state) doesnt acc exist

what is a ponit mutation

change in one amino acid

what mutation causes sickle cell disease and how

Glu6 → Val6

glu is - val is NP → causes it to want to be inside → causes sickling and binding to hydrophobic pocket of other rbcs

explain why each mutation leads to inc or dec in O2 binding affinity

Glu6 → Val6

His F8 → Tyr

Arg141 → His141

His146 → Asp146

sickle cell - glu6 to val6 - sickling due to val being NP and binding to hydrophobic pocket of other RBCs

dec affinity, as His F8 is necessary in binding to Fe2+ in heme so its Fe3+ instead

inc aff, as his is less positive and does not form salt bridges as much, so t state is less stable

inc affinity, as His146 is part of bohr effect and Asp is neg and cannot form salt bridge. without bohr effect and salt bridge, r state is favored.

classes of enzymes: what do they do?

• oxidoreductase

• trasnferase

• hydrolase

• lyase

• isomerase

• ligase

• redox rxns

• transfer function group (ex. kinases)

• hydrolysis reaction

• group elimination to form double bonds

• isomerization

• bond formation coouples w atp hydrolysis

what is the purpose of catalysis

to stabilize transition state, reducing TS energy and making rxn faster

what do the followign do and what does it make at the end

acaid base catalysis

covalent catalysis

metal ion/electrostatic catalysis

1. usses acid or base (H, asp, glu, lys) to transfer/remove a proton from TS to stabilize it, generates CB which can regenerate acid/base with water

2. uses a nucleophilic group (ex. RNH2, so ser, Lys, cys) to form a covalent bond with the substrate to stabilize TS

3. metal ion used to stabilize TS

active site amino acids of chymotrypsin and their role in catalysis

Asp102: stabilizes His57 through H-bonding

His57: general base catalysis

Ser195: covalent catalysis

what does chymotrypsin do and what makes it specific to that substrate?

cleaves polypeptide at the peptide bond prior to the aromatic amino acid

araomatic AA substrates becasue chymotryp has a three AA hydrphobic active site, so it wants large NP substrate, and it has to be a polypeptide because there needs to be a peptide bond

chymotrypsin mechansim

1. His57 acts as a base and removes H from ser195 (-OH) to make it a good nucleophile

2. Ser195 attacks the carbonyl carbon of the scissile (to-be-cleaved) peptide bond) → tetrahedral intermediate 1 w/ oxyanion (C—O-)

3. His57 acts as an acid and donates H to NH (H- -NH). Since NH2 is a good LG. carbonyl C=O reforms, breaking C-N peptide bond to form NH2.

   • the C-terminal half of the polypeptide diffuses away.

   • the N-terminal half of the substrate is covalently stuck to the enzyme via Ser 195 = acyl-enzyme intermediate.

4. His57 acts as a base and takes H from H2O, forming OH-. OH- attacks C=O to form tetrahedral intermediate 2 w/ oxyanion

5. His57 acts as an acid and donates H to Ser195, allowing oxyanion to reform C=O and break Ser195-enzyme bond. → reform enzyme + cleaved polypeptide

how does chymotryp stabilize TS?

how does the oxyanion hole stabilize the oxyanion in tetrahedal intermediates?

4 hydrogen bonds:

3 primary H bonds

1 low barrier h bond

specificty pockets of serine proteases:

• chymotrpsin

• trypsin

• elastase

• chym: 3 uncharged AAs: GSG → likes large NP side chains

• try: neg charge: GDG, D is neg → like pos side chains

• elastase: only two AAs, VT → likes small NP aliphatic (like alanine)

Km = ?

[E total] = ?

[ES] / [E] [S]

[E tot] = [E free] + [ES]

MM eqn: v0 = ?

what is Km? units?

what is kcat? units?

v0 = Vmax[S] / Km + [S]

Km = [S] at V = 0.5Vmax. units = M

kcat = k2 (rate constant for ES → E + P) = rate of turnover. units = 1/s

lineweaver burk eqn: 1/v0 = ?

slope = ?

y int =?

x int = ?

three assumptions of michaelis menten eqn

1. 1 substrate → 1 product

2. k2 = kcat (meanign step 2 is the rate determining step)

3. rate of change of [ES] = 0, i.e. steady state process, rate of formation = rate of depletion

kcat = ?

efficiency = ?

kcat = Vmax / [E tot]

eff = kcat/Km

types of non MM enzymes:

1) ordered sequential

2) random sequential

3) ping pong

1. A + B → C + D, where enzyme E binds to A first, then B, and releases C then D. always in this order

   1. alcohol dehydrogenase

2. A + B → C + D, when E binds to A or B, then the other, and releases C or D, then the other

   1. glycolysis

3. A into E, C out of E, E turns into F. B into F, D out of F, F back into E. \

   1. ex. chymotrypsin

reversible vs irreversible inhibition

reversible: noncovalent - compeititve, noncompetitive, uncompeititve, mixed

irreversible: covalent

how do each of the following affect Km and Vmax?

• compeietive inhib

decribe change in rate/concentraaiton grpah and linweav-burk graph

competitive: increase Km, unchanged Vmax because with enough substrate Vmax can still be reached but affinity/speed of binding is decreased

[S] vs v: graph moves right

LB: slope increases, x int move right. y int the same

how do each of the following affect Km and Vmax?

• noncompeittive inhib

decribe change in rate/concentraaiton grpah and linweav-burk graph

noncompetiitve:

how do each of the following affect Km and Vmax?

• uncompetitive inhib

decribe change in rate/concentraaiton grpah and linweav-burk graph

uncompeititve: inhib only binds to ES not E. Vmax decreases, Km decreases becasue making ES → EI decreases ES which means more E + S → ES, increasing affinity for substrate.

[S] vs v: moves left and down

LB: parallel slope but y-int moves upward, x int moves left

how do each of the following affect Km and Vmax?

• mixed inhib

decribe change in rate/concentraaiton grpah and linweav-burk graph

mixed: inhib binds to both E and ES. Vmax decreases, Km can inc or dec

• alpha < alpha’, ie inhibitor has greater affinity for ES → Km inc (typical)

• alpha > alpha’, ie inhibitor has greater affinity for E → Km dec

[S] vs v: moves right and down

LB: slope increases, y int moves up, x int moves right

Km vs Ki

Km = substrate affinity for enzyme

Ki = inhibitor affinity for enzyme

alpha factor = ? (3 eqns)

Km apparent / Km, when Km app = Km with inhibitor

= 1 + ([I] / Ki)

Vmax / Vmax app

tagret bound meaning does it bind to E or ES

three forms of enzyme regulation:

• substrate level

• feedback inhibition

• covalent modification

substrate level: changing substrate concentration

feedback inhibition: product formation at one step regulates the acitivity of another step

covalent modification:

• reversible: phosphorylation on serine, tyrosine, or theronine (AAs with OH)

of ACTG:

• pyrimidines

• purines

• pairs and # H bonds

pyr: CT

pur: AG

pairs: AT (2 H bonds), CG (3 H bonds)

how does ATCase respond to ATP? CTP?

allosterically

ATP: decreases Km

CTP: increases Km

differences between DNA and RNA

double vs single stradned

thymine vs uracil

deoxyribose (2 OH groups) sugar vs ribose sugar (3 OH groups)

difference between deoxyribose and ribose

ribose - 2’ OH

deoxy - 2’ H → more stable due to not being susceptibel to hydrolsysis

nucleotide vs nucleoside

nucleotide: has O—phosphate (ex. adenoside mono phosphate)

nucleoside: has OH group instead (ex. adenoside)

what is a zymogen

an enzyme that requires a biochemical change to become active, usually by cleavign a certain peptide bond.

• ex. Chymotrypsinogen → Chymotrypsin due to peptide bond cleavage by Trypsin

what three structures form a nucleic acid unit

• sugar (deoxy/ribose)

• phosphate group

• nucleotide (ACTG)

DNA:

• what is the angle between bp

• how many bp per turn

• rise of helix/distance between bp

• 36 degrees

• 10 bp/turn

• 0.34 A between bp

carbohydrate general formula

simplest carb formula

(CH2O)n

simplest: n = 3, C3H6O3

aldose vs ketose - draw

see green

• aldehyde = O=CRH

• ketone = O=CRR

name the following

most stable form of carb: D or L

D

how to know D or L carbon

1. look at last chrial carbon

2. if OH on right = D (r config). OH on left = L (s config)

draw the L glucose

flip ALL chiral groups and top group - mirror image

what is an epimer? if the regular is D, what is the epimer?

carbons that differ in confirmation at a single chiral carbon

also D

#of epimers possible for a heptamer

4 chiral centers, so 2^4 = 16

Anomer

differs in configuration about the anomeric carbon, which is the one bound to two oxygens (alpha vs beta)

steps of cyclization of carbs, ex glucose

1. OH group on last chiral center uses electrons to attack the aldehyde at top → ring with carbon 6 (nonchiral) left out

2. on each carbon in the ring, if OH was on the right, it becomes down. if OH was on the left, it becomes up

3. the attacked carbon can have OH up or down (racemic mix)

alpha vs beta

1. alpha: if the OH at carbon 1 is in opposite direction to the last carbon, CH2OH

2. beta: if the OH at carbon 1 is in same direction to the last carbon, CH2OH

if noCH2OH group was used in cycle formation → in plane, not up/down:

if D enantiomer:

• OH on carbon 1 = up = alpha

• OH on carbon 21 = down = beta

if L enan: switched, up = beta

two types of pentose ketose: name and sturcutre

ribulose - RR (pictured)
xylulose - RL

3 and 4 are chiral

hexose ketose: name and sturcutre

fructose - LRR

3 4 5 are chiral

pentose aldoses: name and structure

ribose: RRR

Arabinose: LRR

xylose: RLR

six membered aldoses

glucose: RLRR

mannose: LLRR (epimer of glu)

galactose: RLLR (epimer of glu)

pyran vs furan

6 = pyran

5 = furan

is alpha or beta most stable? what is the exception? what is changing between the two called?

beta; mannose; mutarotation

naming carbs

1. alpha or beta

2. D or L

3. fructo or whatever else (based on ketose or aldose and how many members)

4. pyranose or furanose

how to tell reducing vs non reducing suger. whihc can be oxidized?

reducign: has a hemiacetal or hemiketal, formed from :OH attacking carbonyl in aldehyde or ketone

1. idenntify anomeric carbon

2. check if it has OH, ie not involved in linkage

reducing can be oxidized back to carbonyl bc the OH is not involved in linkage

how to number carbons on a ring

start from the CH2OH group sticking out of ring = 6, then 5 is the first carbon from there on the ring, the go around away from the O until u get to 1.

how to name disaccharadies

alpha-D-glycopyranoSYL (1-4) alpha-D-glycopyranose

• if anomeric carbon involved: …-glycopyranoSIDE

naming monosaccharide and D/L and aldose/ketose from a ring

• starting from TWO until FIVE, use convention down = right, up = left

• on the last one, OH is at the opposite position of CH2OH

ketose if CH2OH group attached to C on ring with OH. ketose can have same number of carbons but smaller ring

where cna oxidation happen in glucose and what are the resulting forms called

at carbon 1 in a reducing sugar, turning OH back into C=O → gluconic acid

at carbon 6, CH2OH becomes COO- → glucaronic acid

addition of a phosphate group to a monosacch

what are reduced forms of carbs called and what is reduced?

called -ol for alcohol, ex glycerol, ribitol, xylitol

the aldehyde or ketone C=O is reduced to OH

what monosaccharides and disaccharides can mutarotate?

monosaccharides: anything with 4 or more carbons. 3 carbons cannot form a ring so theres no alpha/beta anomers

disacch: if its reducing

• At least one anomeric carbon is free (it retains a free OH group, making it a hemiacetal or hemiketal) → can open into an aldehyde/ketone (non ring) and re-close into alpha or beta configuration.

homopolysacch or heteropolysacch?

all HOMO: all except chitin are made of D-Glucose, chitin is N-amino whatever

why cant we digst cellulose? what part is not digestable?

dont have enzyme cellulase

cant hydrolyze the linkage (BETA 1-4 between glucose)

GAGs structure

short, rod like linear chains (not branched, not double helix bc the charges repel)

compare linkages in the two polymers in starch

which one is glycogen like what makes it different?

Starch contains both amylopectin, α(1-4) glucose polymer with α(1-6) branches, and amylose, α(1-4) unbranched polymer. Glycogen (animals/microbes) is like amylopectin, but higher MW with shorter and more frequent branch point

structure, linkages, abd binding in cellulose

cellulose is a linear glucose polymer with β(14) linkages.

has hydrogen bonding between linked glucose monosaccharides

glycosaminoglycan - what kind of heteropolysacchride?

proteoglycans

proteoglycan vs glycoprotein

proteoglycan - mostly carb

glycoproteins - 95% protein, small amnt carb, blood group identification (certain carb unit makes certain blood type), Serine/Threonine?

how would you change B blood type so everyone could receive it

cleave the B oligosacchride on the glycoprotein?

tamiflu?

competitive inhibitor for sialic acid, inhibiting neuraminidase and blocking release of viral particles