BIOC A (midterm)

something permanently attached to the protein and required for its function is called a _______

prosthetic group

transient reversible interactions with protein are with ______

ligands

ligand binding sites are _____ to ligand and highly specific

complementry

how are substrates different from ligands?

a substrate interacts with an enzyme and is transformed into a product by binding to catalytic/active site

a ligand is is associated with a separate site of a protein/ enzyme and binds transiently not changing into a product

what is an induced fit

what is an example in hexokinase?

a structural change of the protein that makes a binding site more complementry for a ligand

ex. hexokinase conformational change induced by binding of D-glucose

what are the key ligand biniding concepts (4 of them)

regulation, specific, conformational change, regulation

is oxygen soluble in water?

not really

how can oxygen be transported in the blood if not soluble in water?

oxygen binding proteins with reversible binding so oxygen can be loaded and unloaded

what transition metals are found in O2 binding proteins?

Cu and Fe

what is the issue with having free iron in the body?

prone to ROS production

how is the oxygen binding of iron used in the body without producing ROS?

it is incorporated into a heme as a prosthetic group (permanent and part of function)

heme=

protoporphryin IX + Fe2+

porphryins (ex. heme) have 4 pyrole rings connected by ______(-CH=) bridges and linked into a ______ system (CH=CH)

methine

conjugated

a pyrole ring has what in it?

N, double bonds and X groups

what X groups does heme have?

2 (out of 4) are propionate groups (fatty acids)

how many N atoms bind the central Fe2+ atom?

how many bonds can Fe form?

4

6

if Fe can form 6 bonds and 4 are taken up by N, what are the other 2 coordination bonds?

Proximal His F8 and O2

why cant Fe2+ use up 2 coordination bonds for O2?

it would oxidize to Fe3+ which cannot bind O2

what on the proximal His residue binds to the Fe2+?

the amino (N)

what does coordination bond mean? How many does central Fe2+ form?

using e- form only one atom, 6

3 common globin oxygen binding proteins and their functions

myoglobin- monomer stores O2 in muscle

hemoglobin- tetramer of 2 a globins and 2 B globins acts as O2 transporter

leghemoglobin- found in legume plants as a way to sequester O2 away from O2 sensitive N2 fixing bacteria

myoglobin is a monomer (single peptide chain) with ____ resides weighing _____

153, 16.7kDa

a globin monomer has ____ a helices labeld _____-_____

8 helices, A-H

where is the proximal histidine located? where is the distal histidine located?

F8 (His93), E7 (His64)

what does globular mean?

roughly spherical shape

How are globins named?

A-H by alpha helix and then also N and C terminals.

Connecting loops labeled (ex. AB3- the thrid AA between helix A and B).

how can you tell apart the C helix and C terminal in naming?

anything labeled HC__ indicates C terminal, as H is the last helix and all AA would be between the H helix and C term if labeled HC.

the C helix is reffered to with C__, BC__ or CD__

N is a helix in a globin (T/F)

F, there is no helix N, N represents the N (amino) terminal

the heme binding pocket of myoglobin (or a subunit of hemobglin) is formed by what helices?

E and F

where are propionate side chains of heme in globin ?

the surface

heme has 2 polar histidine residues near it, what is the rest of the heme surrounded by?

non-polar AAs

what is the role of histidine F8 (5th coordination bond of Fe2+ in heme)

directly bonded to Fe2+ as proximal histidine preventing oxidation

what is the role of the distal histidine E7?

close but not bound to central heme Fe2+, rather acts as an enhancer of oxygen binding (oxygen forms 6th coordination bond with central Fe2+ and hydrogen bond with E7)

F8 and E7 are conserved in all globins (T/F)

T

how does oxygen enter Mb? (or Hb subunit)

proteins do “breathing” where 1A molecular motions of AA side chains in nanoseconds move just enough for O2 to slip in and bind to Fe2+ and E7

how can you measure o2 binding of globins using visable light?

the conjugated bonds have strong visable light absorption and O2 binding effects the electron distribution- changing light absorption

oxy-heme found in arterial blood abosrbs ____ appearing _____

deoxy-heme found in venous blood absorbs _____ appearing ____

blue, red

red, blueish

Ka=

Ka=[PL]/[P][L]

the association constant

Kd=

Kd= [P][L]/[PL]

the dissociation constant

Ka is equal to the reciprocal of Kd and vice versa (T/F)

T

Kd=1/Ka

Ka=1/Kd

what are Ka and Kd used to describe

ligand binding (reverisbility, strength, inhibition)

what is Ka and Kd bad at describing?

O2 binding characteristics

if Kd is high affininity is ____

low (higher Kd means more substrate to reach ½ Vmax)

is Y (occupancy) ever less than 1?

no

what are the two terms used interchangeably for occupancy?

Y and theta

for non O2 binding: Y=

Y= binding sites occupied/ total binding sites

Y=[PL]/ [PL]+[P]

Y=[L]/[L]+Kd

for a ligand binding curve, Y is plotted as a ________ function of [L]

hyperbolic

why is [L] in equation diff than [L] on x axis?

[L] on x axis is the total ligand, [L] in equation is only the free ligand

with globins (O2 binding) Y=

Y=pO2/ pO2+p50

define pO2 and p50

pO2 is the partial pressure of oxygen

p50 is the partial pressure of O2 when ½ Vmax

(use partial pressure instead of concentration with O2)

for O2 binding proteins the graph of Y as a function of pO2 is ________

hyperbolic

why can Kd take on so many values (from 10^-16 to 10^-2)

in the body proteins need a wide variety of affinities to preform various functions

CO binds free heme ______X greater affinity than O2

CO when in Mb/Hb subunit heme binds ____X greater affinity thean O2

20 000X

200X

why is CO less favoured in heme in protein than free heme?

distal His E7 creates steric hinderance with CO, and a favourable H bond with O2

O2 binds heme____ Co binds heme____

at an angle, straight upright

why is Mb bad for transport and better for storage?

Mb is insensitive to pO2 change in body, at tissue and lung pO2 Mb is fully saturated with O2 and bidns with high affinity, the curve on a graph (Y as function of PO2) is hyperbolic

____ structure of Hb and Mb look similar

tertirary (a heme subunit)

F8, E7 histidiens and ____ are highly conserved between globins

F4 Leu

out of 150 AA ___% are identical between Mb and Hb subunits.

Is this a lot?

18%

its not extraordinary, most proteins have high similarity so its actually surprising that it isn’t more as they have similar functions.

Mb is found in vertebrate blood (T/F)

F

what is the major O2 carrier in vertebrates?

Hb

what is Hb size?

64.5 kDa, 4 tetramers of polypeptides with 1 heme each

human adult Hb 2a (141AA) and 2B (146AA) subunits in Hb, where can the strongest interactions be found?

a1-B1, a2-B2 (about 30 residues)

(a1-B2, a2-B1 only 19 residues)

what types of interactions hold together units of Hb?

hydrophobic effects, H-bonds, ion pairs (salt bridges)

what is the layout of the Hb subunits?

a2 B1

B2 a1

is Hb an enzyme?

naur

what would be fatal if we had free heme?

standing near a road (CO poisioning)

at which subunit interfaces of Hb do important ion pairs occur? what are these pairs?

a1-B2 and a2-B1

B1 subunit His HC3 forms salt bridges within the B1 subunit at Asp FG1 and with the a2 subunit at Lys C5

(B1 Asp FG1 —-B1 HisHC3—-a2 Lys C5)

what is pO2 in lungs?

pO2 in tissues?

13kPa

4kPa

what affinity does Mb bind O2?

high affinity, P50=0.26 kPa

what affinity does Hb have for O2?

high in lungs, low in tissue, apparent p50= 3.5kPa but changes with structure changes

what is the high affinity state of Hb? what is low affinity state?

high- R low-T

what is an allosteric protein

a protein where binding of one ligand effects binding properties of another site

is Hb allosteric? what about Mb?

Hb is allosteric (notice sigmoidal binding curve)

Mb only has one O2 binding site as is is a monomer so no it is not allosteric (hyperbolic bindng curve)

what is a homotropic allosteric modulator?

what is a heterotropic allosteric modulator?

homo- modulator is a ligand (binds at ligand binding site)

hetero- modulator is not a ligand (binds at different site)

are modulators activators or inhibitors?

can be both

in Hb O2 a ligand, but it also acts as a _______ (neg or pos) _______(hetero or homo) modulator

positive homotropic

in which state of Hb is F8 and heme porphryin ring puckered?

T

P50 changes in Hb depending on O2 presence (T/F)

T

binding of oxygen to Hb has what effect on the heme structure?

T→R

with O2 binding Fe2+ is re positioned within the heme, pucker is reduced on porphryin ring which pulls the F8 His and the F helix adjusts triggering T→R

what ion pairs break in T→R in Hb?

B1 Asp FG1 —-B1 HisHC3—-a2 Lys C5

what state do the B1 Asp FG1 —-B1 HisHC3—-a2 Lys C5 ion pairs stablilize?

T

the R state no longer has any salt bridges (T/F)

F- while the B1 Asp FG1 —-B1 HisHC3—-a2 Lys C5 ion pair is broken there are still salt bridges that exist within the protein

when oxygen is released into tissue what % of Hb oxygen capacity is released?

38%

myoglobin acts as if it is in a permanent ___ state, having high affinity for O2 in lung and tissue.

R

a protein with a high or low hyperbolic curve is allosteric (T/F)

F, only sigmoidal curves show allostery

P+L → PL only works for when you have one binding site (ex. Mb) but with multiple

what is the hill equation?

log(Y/1-Y)=nlogpO2-nlogP50

model the binding of ligands to proteins as a function of concentration, specifically describing sigmoidal curves

what is nH?

degree of cooperativity (hill coefficient)

nH <1 means ____

nH=1 means _____

nH>1 means _____

nH <1 means neg cooperativity

nH=1 means no cooperativity

nH>1 means pos cooperativity

what nH would Mb have?

nH=1 (no cooperativity)

describe what concerted means

all subunits are either in one state or another, they all shift together simutaneously

what model describes when conformation changes independantly in subunits when ligans bind

sequential

what is n in equations?

the number of binding sites for the ligand ex. n=4 for Hb

what is another name for the concerted model

MWC

Hb transports H+ and CO2 as well as O2.

do H+ and CO2 bind to the Oxygen binding site?

no they each have their own sites

what is the relationship between O2 binding and H+/Co2 binding?

inverse, negative heterotropic allostery (when H+/CO2 bind, O2 is more wanting to be released)

why is CO poisioning worse than anemia if in both cases Hb can only bind 50% O2?

CO prevents offload of O2 into tissues and is strongly bound to Hb showing a hyperbolic curve. with anemia O2 can still offload into tissues as seen with sigmoidal curve

what is the effect of CO2 and H+ on O2 binding called?

Bohr effect

describe pH in tissues vs lungs

low pH in tissues (high [H+]) and high CO2- causes O2 to unload (T state)

higher pH in lungs, CO2 and H+ excreted and O2 binds (R state)