Download Structural Properties of Enzymes

The Structure of Enzymes
1. Determination of Mr
Molecular mass (MW) has the dimension,
Dalton (Da). Mr is defined as a dimensionless relative molecular mass
(molecular mass of a given molecule divided by the mass of 1/12 of one atom
of 12C (1.66 x 10-24g)). Mr of a purified protein can be determined by either:
a. Ultracentrifugation
Analytical ultracentifuges are equipped with
a window which allows the monitoring of protein bands
(spectrophotometrically) as they move radially with application of
centrifugal force. The rate of movement of these bands, the rate of
diffusion (widening of the band), and the point at which the bands quit
moving can be measured and molecular mass can be determined
according to equations set forth initially by Svedberg. When dissolved
in aqueous (or other) solvents, enzymes stay in solution because
solvation energy (ΔGsolv), which is determined by the solvent
accessible surface area is greater than gravitation force. High
centrifugal forces can exceed ΔGsolv, for large molecules such that
under the influence of such forces, large molecules tend to sediment.
Molecules with larger Mr sediment faster such that the rate of
sedimentation (v) can be used to determine the Mr, as long as certain
other physical properties of the protein such as its diffusion constant
(D, empirically determined in an analytical ultracentrifuge at very low
centrifugal force), partial specific volume (, determined by measuring
the volume change upon addition of 1g of a substance to a large
volume of solvent), and the density of the solvent (empirically
determined) are known. The equation describing the relationship of
Mr to sedimentation velocity, D, and  is as follows (see derivation):
Mr = RTs/D(1-),
R = gas constant, T =temp (K), and s = Svedberg constant. 
As these molecules sediment towards the bottom of the
centrifugal chamber (centrifuge tube), at some point, the centrigual
force is balanced by the buoyant force, tendency of these molecules to
diffuse away from the high concentration locus at the bottom of the
tube, and friction. If centrifugal force is not sufficiently strong, the
protein will stop moving after sometime, after the sedimenting and
opposing forces are equal. This condition, called sedimentation
equilibrium can be described by the equation (see derivation):
Mr = (2RT/(1-))* dln(c)/dr2
Where c is the distribution of concentration of protein within
the band as a function of distance (r) along the cell (distance from the
center).
b. Gel filtration
The rate at which a protein traverses a size
exclusion column is inversely proportional to its size. Very large
proteins exit first, and the smallest molecules last. The Ve (elution
volume of the protein of interest), Vo (elution volume of very large
molecule(s) completely excluded by the gel), Vi (the elution volume of
a very small molecule which is maximally retarded by the gel), define
the Kd, the distribution coefficient for a given column.
Kd = (Ve – Vo)/ (Vi - Vo)
A plot of Kd vs. the Mr of standards will yield a curve
which is roughly linear decreasing from Kd ~1 for very
small proteins, to Kd ~0 for very large ones (see figure)
1
0.8
Kd
0.6
0.4
0.2
0
3
4
5
6
log (Mr)
c. SDS-PAGE
The rate of migration in an SDS-PAGE gel is
inversely proportional to the size of the protein, such that log (Mr) is
inversely proportional to the distance traveled in the gel (see chapter
2).
d. Mass Spectrometry
FAB-MS (Fast Atom Bombardment-Mass
Spectrometry) is a relatively new technique in which the protein of
interest in dissolved in glycerol, and bombarded with high energy Xe
or Ar atoms. The gas-phase ions thus created are propelled in
electrical and magnetic fields proportional to their charge and mass.
Mr of peptides with up to 50 aa can be done with FAB-MS.
MALDITOF (Matrix Assisted Laser Desorption Ionization Time of
Flight) mass spectrometry uses intense pulded UV laser beams to
vaporize small samples of proteins dissolved in organic matrices (such
as cinnamic acid). The resulting ions are accelerated to defined kinetic
energy before striking the detector. This technique can resolve Mr of
proteins with MW > several hundred thousand. In ESI (ElectroSpray
Ionization) mass spectrometry, the protein of interest is dissolbed in
formic acid and methanol/acetonitrile and pumped through a charged
capillary needle with 4 kV potential, resulting in a fine spray of ions
from which the solvent molecules evaporate, leaving the ion alone. A
quadrapole mass analyzer operated at low pressure (10-8ATM) is used
to determine the charge/mass ratio. ESIMS is capable of giving the
intact molecular mass of an oligomeric protein.
2. Determination of Amino Acid Composition
a. Amino Acid Composition
i. Amino Acids
L-stereoisomers are used. There are 7
nonpolar, 7 polar uncharged, 2 acidic (polar, charged,
negative), and 3 basic (polar, charged, positive). See
abbreviations and codons.
ii. Peptide Bonds
Amide bonds formed between the Cterminal of the first amino acid and the N-terminal of the next.
iii. Peptides
Chains of amino acids linked by peptide
bonds, conventionally written beginning with the amino acid
which has the free amino group (N-terminal)
iv. Disulfide bonds Formed between cysteine residues (residues
is another term for an amino acid present in a peptide) of two
separate peptides, or within one peptide.
v. Hydrolysis of amide bonds:
1. 6 N HCl, reagent grade at 383 K for 24 h in a vacuum
(near total loss of tryptophan, and partial (10%) loss of
serine, threonine, and tyrosine
2. p-toluene-sulfonic acid – preserves tryptophan
3. Alkaline Hydrolysis
NaOH – preserves tryptophan
4. Alkylation of cysteine followed by hydrolysis by
methanesulfonic acid in the presence of tryptamine
which serves as an amino-protectant.
5. Peptide bonds between branched-chain amino-acids
(i.e. Ile-Ile, Ile-Val etc.) are less susceptible to acid
hydrolysis, and may require a longer time for
hydrolysis.
6. Asparagine and glutamine are hydrolysed to aspartic
and glutamic acid.
7. Cysteine, cysteine and methionine can be reliably
determined by performic acid oxidation prior to
hydrolysis. The oxidized derivatives are easily
measurable.
vi. Analysis of amino acid mixtures
1. Ion-exchange column (sulfonated polystyrene); elution
with stepwise gradient from 2.2 to 5.3. Acidic amino
acids elute first and basic last.
2. The amino acids eluted from the column are reacted
with ninhydrin to yield purple colored adducts (with
exception of the adduct with proline, which is yellow)
followed by spectrophotometric detection.
3. Adducts of amino-acids with fluorescamine or
pthalaldehyde are highly fluorescent, thus detectable at
pmol levels.
4. Derivatives or amino-acids with pthalaldehyde are more
stable than of fluorescamine, but proline must first be
reacted with alkaline hypochlorite to allow reaction
with pthalaldehyde.
b. Uses of amino-acid composition analysis
i. If the primary structure of a given protein is known, the degree
to which the amino-acid composition analysis approaches the
predicted amino-acid composition is a measure of purity.
ii. Predictions of general properties of a protein on the basis of
predominance of specific amino acids (i.e. known proteins
which are characteristically rich in particular amino-acids, such
as lysine, glutamic acid, proline, cysteine, tryptophan etc.,
often have characteristic structural/functional properties).
3. Determination of Primary Structure
a. Indirect method: More frequently utilized now. If you can sequence
a small fraction of the purified protein, you can use the sequence
imformation to clone the protein from a cDNA library, and determine
the amino-acid sequence from the nucleotide sequence of the clone.
b. Direct method:
i. 10-100 nmol enzyme
ii. Edman technique based sequential derivatization of the Nterminal amino acid of an immobilized peptide, followed by
chromatographic identification of the derivative.