TA BIOCHEM Review Flashcards
isoelectric point (refers to the whole protein)
-pH at which the net charge of the amino acid is zero
-ALSO roughly 50% aa’s deprotonated and the other 50% are protonated
when pH below the pl the overall charge of the protein is
positive (think H+ acid)
when pH above the pl the overall charge of the protein is
negative (think OH-)
(A-/HA)= 10^(+) or value>1
DEPROTONATED
(A-/HA)= 10^(-) or value<1
PROTONATED
gel filtration chromatography or size exclusion chromatography
larger molecules elute first whereas smaller molecules elute later
ion exchange chromatography has 2 types
-DEAE (weak anion exchanger)
-CM (weak cation exchanger)
ion exchange chromatography interaction strenght with exchange matrix depends on:
-charge density of protein (modulated by pH)
-strength of ions that compete with protein for binding
2 ways to elute
- modulate pH (start with getting protein with most basic pH a positive charge and wash, repeat to get all of the through)
- increase salt concentrations (both +/- interactions) -> will compete for binding and displace bound proteins with smaller overall charge first ie pl closer to the pH e.g. have 3 protein pH’s of 3,5,7 C would wash away first if using a pH of 9
SDS ONLY reducing
reduces disulfide bridges so same protein appears as smaller in reducing but as whole size in non-reducing
delta H>0
when BREAKING bonds
delta H<0
when FORMING bonds
S>0 and H<0
spontaneous at ALL temps (G<0)
S<0 and H<0
spontaneous at low temps (when TdeltaS is small)
H>0 and S<0
spontaneous at high temps (when TdeltaS is large)
S<0 and H>0
non-spontaneous at all T (G>0)
hemoglobin and hemoglobin have similar binding sites BUT
different quaternanry structures
Fe(II) octahedrally coordinated by four nitrogen groups of poryohrin ring, O2 and His F8
Hb
Deoxy-Hb (T-state) -> RELAXED
-energetically favored when NO oxygen
-lower pH stabilizes with salt brideges
-BPG binds central cavity and increases favorability to stay in T-state
Oxy-Hb (R-state) -> TIGHT
-energetically favored when THERE IS oxygen
Cooperativity in Hb
-O2 + Fe(II)
-Fe(II) moves into plane of heme
-HisF8 interacting with Fe(II) pulled up like a lever and moves helix F
-surronding subunits move relative to one another and change pKa of certain amino acids involved in salt bridges needed to stabilize T-state
-protons released in these amino acid groups and T-state salt bridges break (because pH inc.)
-the cavity in the center shrinks but the C2 symmetry remains in the R-state
cooperativity O2
favors R-state
cooperativity H+
favors T-state (dec. pH) RIGHT shift
cooperativity CO2
favors T-state (dec. pH) RIGHT shift
cooperativity BPG
favors T-state (high altitudes more tightly bound) RIGHT shift
cooperativity CO
favors R-state (competitive inhibitor) LEFT shift
what type of curve does Hb have?
sigmodial
what type of curve does myoglobin have?
hyperbolic
Hb exhibits what type of cooperativity?
positive :D
cholera toxin
-hetero-hexamer
-C5 cyclical point symmetry
-B-pentamer + A-subunit = AB5
globular actin (G-actin)
-4-domain subunit
-helical symmetry
GroEL.GroES
-CHAPERONE
-C7 symmetry in subunits
-NOT D3 symmetry in upper/lower rings because conformational changes
-involves ADP binding
ATCase
-D3
-12 total chains
-6 regulatory chains (R-chains) -> binding of CTP to R subunits causes R-> T which INHIBITS catalysis
-6 catalytic chains (C-chains) -> binding of substrate to the catalytic subunits causes T->R which causes CATALYSIS
ATCase catalyzes the first committed step in
pyrimidine synthesis (CTP & UTP)
myoglobin
-8 alpha helices
-ONE subunit
-Fe in center
Hb
-4 total subunits
-2 alpha sub units -> C2
-2 beta sub units -> C2
-Fe in center
-cooperativity
hemocyanin
-D3
-Cu in center
-THE METAL IS OXIDIZED UNLIKE HB
calmodulin
-Ca2+ dependent conformational change
TIM
beta-BARREL surrounded by alpha helices
chymotrypsin
cleaves large hydrophobic chains -> Tyr, Typ, Phe, Leu
Trypsin
cleaves Lys or Arg
elastase
cleaves ala, gly, val
All (chymotrpysin, trypsin, elastase) cleave
peptide bond of amino acids
ribonuclease A
-BETA protein with FOUR disulfide bonds and four ANTI-PARALLEL strands
-RNA binds 2 His in active sire, Lys residue to stabilize phosphate group
-endonuclease that cleaves single-stranded RNA (P-O bond)
-secreted from pancreas and cleaves in digestive tract, no specific sequence
neuramindiase
-PROPELLER
-4 beta strands per propeller
-2 Glu in active site
-cleaves sialic acid from cell surface
-facilitates virus release from infected cells
keratin
-a-b-c-d-e-f-g psuedo pattern
-two right-handed helices form left-handed coiled structure
collagen triple helix
-2 alpha-1 and alpha-2 chains (NOT related to 2° structure)
-RIGHT handed twist
-Glo-Pro-Hyp (HYDROPHOBIC) repeats
k1 -> ES
BUILDUP of ES
k-1 <- ES -> k2
BREAKDOWN of ES
Km
=micheaelis constant
=[S] at 1/2 Vmax
=(k-1 + k2)/k1
the Km/Vmax in the equation 1/Vnaught=Km/Vmax x 1/[S] + 1/Vmax represents
SLOPE
the 1/Vmax in the equation 1/Vnaught=Km/Vmax x 1/[S] + 1/Vmax represents
INTERCEPT
Vnaught=
Vmax[S] / Km + [S]
assumptions of MM
- only Vnaught is measured
- steady-state assumption
- [E] «_space;[S]
competitive inhibitor
-alpha changes in both equations (Lineweaver-burk and MM)
-ONLY Km changes
-inhibitor binds to enzyme before substrate
uncompetitve inhibitor
-alpha’ changes
-changes BOTH Vmax (lower) and Km
-PARALLEL LINES
-slower rate because binds to allosteric site -> unable to catalyze substrate -> lower Vmax
mixed/non-competitive inhibitor
-BOTH alpha & alpha’ changes
-slopes, x/y-intercept are ALL different
-x-intercept has both a’ and a contributing to its changes
-Km ONLY unaffected is a=a’
Catalytic mechanisms
- acid-base
- metal ion
- catalysis via proximity and orientation
- covalent catalysis -> nucleophiles and electrophiles
- catalysis by preferential TS binding
carbonic amhydrase (metal ion catalysis)
-Zn2+ polarizes a water molecules which ionizes due to the action of a fourth (not shown) His and becomes OH-
-OH- performs nucleiphillic attack on the C atom of the CO2 substrate
-HCO3- product
-H2O comes in and cycle repeats
Serine proteases are a mixture of
-covalent catalysis
-concerted acid-base catalysis
-TS stabilization
Serine proteases functions and characteristics
-Asp102 orients His57
-His57 = general acid/base
-Ser195 forms covalent bond with peptide to be cleaved
-covalent bond formation turns a trigonal C into a tetrahedral C of intermediate
-tetrahedral oxyanion intermediate stabilized by main NHs of Gly193 and Ser195-> OXYANION HOLE