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Structure of
Enzymes
OUTLINE
• Determination of relative molecular
mass, Mr
• 1O structure and amino acid
composition
• 2O and 3O structure (3-D structure)
• 4O structure (arrangement of
subunits)
Relative molecular mass, Mr
(molecular weight)
• Mr is a dimensionless number: the ratio of the molecular
mass of a molecule to 1/12 the mass of the atom of 12C
• Ranging from 10 000 to several million
• Methods:
1. Ultracentrifugation
2. Gel filtration
3. SDS-PAGE
4. Mass spectroscopy
Ultracentrifugation
• 65 000 rpm ~ 300 000 x gravity
• Sedimentation velocity: sedimentation can be monitored
(refractive index or absorbtion at 280 nm) and sedimentation
coefficient (s) can be determined
RTs
Mr =
D(1-v)
D: diffusion coefficient, : density of solution
v: partial specific volume, R: gas constant
T: temperature
• Sedimentation equilibrium: In stead of complete sedimentation, an
equilibrium state is reached
– The advantage is that the system is studied in equilibrium so there is no
dependence on the shape of solute or the viscosity of solution
Gel filtration
• Simple but Mr found should only be used as a guide (problems with
non-globular proteins and glycoproteins)
• For globular proteins, 5-10 % accuracy can be obtained
Sodium dodecylsulphate polyacrylamide gel
electrophoresis (SDS-PAGE)
Mr found by this method should be checked
by other methods
• Histones are highly (+)ly charged
• Glycoproteins may show impaired binding
of SDS and display low affinities
• Subunits?
Mass Spectrometry-1
the most recent and sensitive technique...
• Mass Spectrometry is a method for determining the mass of
molecules by producing and analyzing charged species (ions)
• In the late 1980s, “soft” ionization techniques allows the production of
ions of low energy and facilitated the ionization of large biomolecules
• Good sensitivity (0.01-0.002 % accuracy)
– Protein modifications and post-translational processing can be detected
– Mass change induced by site-directed mutagenesis can be confirmed
• Earlier techniques such as SDS-PAGE produce a mass accuracy in
the range ± 5 %
Mass Spectrometry-2
• Matrix-assisted laser desorption ionization-time of flight
(MALDI-TOF)
Dried
protein sol’n
+ org. matrix
matrix
absorbs UV
evaporizes and
carries protein ions
into gas phase
ions are
accelarated
to the detector
– MALDI-TOF is particularly suitable for high molecular mass proteins (>
250 000)
– Good tolerance to biochemical buffers and salts
– In the analysis of mixtures
– Fast
– Lower accuracy
Mass Spectrometry-3
• Electrospray ionization (ESI)
Protein +
volatile org.
solvent
thru a capillary
needle maintained
at 4kV potential
fine spray solvent evaporated
of small
and ions are
droplets with
formed
high surf. charge
– Sensitivity deteriorates with the presence of non-volatile buffers and
other additives
– It is more complex, more difficult and therefore more expensive than
MALDI-TOF analysis
– Mass up to 100 000 with 0.002% accuracy
– Can interface with HPLC  peptide mixture separated in LC column
can be directed to MS
Primary Structure-1
• The structure and reactivity of a protein are defined by the
properties and order of amino acids constituing it
• In proteins, amino acids are identified numerically, starting with the
N-terminus
• Two amino acids condense to form a amide or peptide bond 
polypeptides
60 % C=O
40 % C=N
Primary Structure-2
Amino acids
• Most of the enzymes are proteins made up of amino acids (20)
• Additional components such as metal ions, cofactors or
carbohydrates may be present
• General formula: (R: side chain and Central C atom: alpha carbon (C))
+NH
3-CH-CO2
(except for proline) zwitterion form
R
• Except for glycine, all the amino
acids have a chiral center, so
they can exist in more than one
enantiometric form, L and D-form
• In enzymes, all amino acids
occur in the L-form
A. Acids
20 + 2 proteinogenic a.acids
• Selenocystein (UGA)
• Pyrrolysine (UAG) (in
methanogenic archea and in
one bacteria, related to
methane metabolism)
Primary Structure-3
Properties of a.acid side chains:
Hydrophobicity: (valine, leucine, alanine, etc)
• They tend to cluster in polar solvent  hydrophobic interaction
 Strong driving force to form the 3-D structure....
 Form hydrophobic core, help to stabilize nonpolar substrate binding
Hydrogen bonding:
• H-bonding btw. side chain and peptide backbone help to stabilize the
structure
• H-bonding btw. side chain and ligands can contribute to ovarall binding
energy
Salt-bridge formation:
• non-covalent electrostatic interactions btw electronegative and
electropositive species in proteins
 Protein-nucleic acid interactions (usually lysine and arginine)
 Cytochrome c (lysine region)-cytochrome oxidase (aspartic and glutamic acid
region)
Primary Structure-4
A. acids as acids and bases:
• Tyrosine, histidine, cysteine, lysine, arginine and aspartic and
glutamic acids (have titratable protons)
 Proton transfer to and from reactant and product molecules
 Nucleophilic and electrophilic reactions with reactant molecules  bond
cleavage or formation
Cation and metal binding:
• Many enzymes incorporate divalent cations (Mg2+, Ca2+, Zn2+) and
transition metal ions (Fe, Cu, Ni, etc)
 Stabilize their structure (e.g. zinc center of insulin)
 Redox centers for catalysis (e.g. heme-iron centers)
 Electrophilic reactants (active site zinc ions of metalloproteases)
• Imidazole ring of histidine is particularly important
Primary Structure-5
Covalent bond formation:
• Disulfide bonds: btw two cysteine residues, sulfur-sulfur bond
– Espacially important to stabilize the structure
• Phosphorylation: kinases vs phosphatases
– OH groups of threonine and serine (most common)
– Greatly affect the biological activity
• Glycosylation:
– Sugars attached to OH groups of threonine and serine (O-linked) or at
the nitrogen of asparagine side chain (N-linked)
– Solubility, folding and biological reactivity is affected
Primary Structure-6
Amino acid analysis
• Peptide bond: for hydrolysis: in HCl, at 110OC, 24 h under vacuum
• Disulphide bond is the only other type of covalent bond in enzymes;
they can be broken by 2-mercaptoethanol or dithiothreitol
• Classic a.acid analyzer: an ion-exchange column (Stein and Moore,
1960s)
– A.acids are eluted by a buffer of increasing pH (2.2 to 5.28)
– Eluents are analyzed by ninhydrin analysis (570 nm, except for proline:
440 nm)
– Fluorescamine or phthalaldehyde can also be used
• HPLC can also be used for analysis of a.acid mixtures
• Some properties can be determined by a.acid analysis:
–
–
–
–
High non-polar a.acid content: high content of helical structure
Useful in calculation of partial specific volume
Concentration determination of a purified protein
etc
Primary Structure-7
Determination of primary structure
• Direct method: operates at protein level (1953, Sanger),10-100
nmol enzyme is enough
–
–
–
–
–
Cleavage of polypeptide chain (proteases, some chemical reagents)
Separation of the fragments (by size, charge, etc)
Sequencing of the purified peptides (Edman degradation method)
Preparation and analysis of new fragments
Alignment of peptide sequences and determination of overall sequence
• Indirect method: operates at DNA level (1970s)
– Small amount of sequence information is needed
• Penicilinase from E.coli:
– direct method: 4 years
– indirect method: 6 months and also resolved some of the ambiguities
encountered in the direct method
Primary Structure-8
• Sequence information is useful....
–
–
–
–
–
–
To calculate the Mr of an enzyme (validation of other results)
To locate a particular amino acid in an enzyme
To interprete data from X-ray crystallography
To predict the 3-D structure
To identify functional regions
To explore relationship between enzymes
• Degree of similarity between enzymes
European Bioinformatics Institute (EBI) provides BLAST, FASTA and BLITZ
• Establishment of evolutionary relationships between organisms
Phylogenetic tree (TREE and PHYLIP)
Secondary structure-1
Secondary structure
• Primary structure of an enzyme do not
explain the catalytic power and specificity of
the enzyme
• How polypeptide chain is folded up to bring
different parts together is important
– Binding sites are created
– Unusual environments for catalysis is
created
• Some characteristics of peptide bond is
important to understand the 2O structure
– Cis-trans forms
– Delocalisation of electrons
Secondary structure-2
• Delocalization of the peptide π
system restrict rotation about the
C-N axis
• When possible rotations in different
axis are analized, it was seen that
(except for glycine) two pairs of
angles are common in proteins
– Right-handed  helix
– -sheet
 Highly regular local substructures
Secondary structure-3
Right-handed  helix
• Most commonly found protein secondary structure
• Structure is stabilized by a network of H-bonds btw
– carbonyl O of residue “i”
– nitrogeneous H of residue “i + 4”
• Side chains of a.acids all point away from the axis  minimize steric
crowding
• Each turn:
– 3.6 a.acid residue
– 5.4 Å (1.5 Å per residue)
• Network of H-bond eliminates the interaction of these groups with
polar solvents
– In membrane bilayer, -helical structure tend to form (ca 20 a.acid in
this form is needed)
• Proline act as a helix-breaker
Secondary structure-4
-sheet
• It is composed of fully extended
polypeptide chains linked together
through H-bonding btw adjacent
strands
• Both intermolecular and
intramolecular -sheet is possible
Secondary structure-5
-turns
• Reverse turn, hairpin turn or -bend
• Short segments of the polypeptide chain that allow it to
change direction
• Turns are composed of 4 a.acids in a compact configuration
• Proline and glycine are generally found in the turns
Random coil
• Well-defined secondary structures are interspersed with
regions of nonrepeating, unordered structure known as
random coil
• This provide dynamic flexibility to the protein
 Facilitate the biological activity
Secondary structure-6
Supersecondary structure
• Intermediate to secondary and tertiary structure
• Levitt and Chothia (1976) classified proteins on the basis of
structural comparisons
– proteins containing mostly -helix
-proteins
– proteins containing mostly -sheet
-proteins
– proteins that contain -helices and -strands in an irregular sequence
 + -proteins
–  /  proteins with alternate segments of -helices and -strands
/-proteins
Tertiary structure-1
Tertiary structure
• Arrangement of secondary structure elements and a.acid side chain
interactions that define the 3-D structure of the protein
• Folded structure of protein
• Folding process is remarkable since under the right conditions, it will
proceed spontaneously in vitro
• Results of proper folding:
– Hydrophobic residues are buried away from polar solvent
– Distant a.acid side chains can interact with each other (chemically
reactive centers are formed)
– Folds or pockets which can accomodate small molecules are formed
– In some proteins, discrete regions of compact tertiary structures that are
seperated by more flexible regions can be found (domains)
Tertiary structure-2
Different representations
Stick
diagrams:
shows the
position of the
atoms in space
Spacefill
the atoms are
presented as merged
spheres that
approximate their
Van der Waals radii.
this gives an idea of
the volume occupied
by the molecule.
Ball and stick
Atoms are
represented as
small spheres
(NO relationship
to the size of the
atoms)
Cartoons
(ribbon diagram)
it is common to
represent
structures as
cartoons, where no
bonds are shown
at all.
Ferredoxin reductase
porphobilinogen deaminase
Heme-dependent catalase
Secondary and tertiary structure
Determination of structure-1
• 1946 Nobel – J.B. Sumner - proteins can be crystalized (in 1926,
urease)
• 1962 Nobel - M.F. Perutz and J.C Kendrew - structure of globular
proteins (in 1957, myoglobin)
• 1964 Nobel – D.C. Hodgkin - for her determinations by X-ray
techniques: the structures of important biochemical substances
(1937-cholesterol)
• 2002 Nobel - K. Wüthrich - for his development of nuclear magnetic
resonance (NMR) spectroscopy for determining the 3-D structure of
biological macromolecules in solution (80s)
Secondary and tertiary structure
Determination of structure-2
X-ray crystallography
• By far the most widely applied technique
• Scattering of electromagnetic radiation of suitable wavelength by
electrons belonging to the atoms in a molecule
• Basics:
– Crystals are three dimensional ordered structures. The 3D location of
atoms within a unit cell can be listed as their x, y, z Cartesian
Coordinates.
– Visible light has a wavelength much longer that the distance between
atoms so it is useless to see molecules. In order to see molecules it is
necessary to use a form of electromagnetic radiation with a wavelength
on the order of bond lengths, such as X-ray
– Electrons diffract the X-rays, which causes a diffraction pattern. Using
the mathematical Fourier transform these patterns can converted into
electron density maps.
– To get a 3-D picture, the crystal is rotated to get 2-D electron density
maps for each angle of rotation. Computer programs needed to find the
3-D spatial coordinates.
Secondary and tertiary structure
Determination of structure-3
electron density map
To interprete a electron density map
of an enzyme, it is necessary to
know a.acid sequence, then side
chains can be located
Secondary and tertiary structure
Determination of structure-4
Basic requirements for X-ray crystallography
• Crystals of enzyme: 0.1 mm or larger, reasonably stable
• Isomorphous heavy-atom derivatives: Incorporation of heavy
atoms must not distort the structure  information on phases
• Computing facilities:
– To calculate the electron density maps
– To refine structure
– To display the structure
• Primary structure: to interpret the electron density map in terms of
covalent structure of the molecule
Secondary and tertiary structure
Determination of structure-5
Enzymes in solution? Time-average of various conformational states...
Nuclear Magnetic Resonance (NMR)
• Determination of the structure depends on measuring the strength of
interactions btw different nuclei  distance is calculated
• For enzymes < 25 kDa
• Relatively high concentrations of enzyme required (1 mmol/L)
Circular dichroism (CD)
• Based on the interaction of circularly polarized light with chiral
(optically active) compounds
• More limited structural info can be obtained
 Time average structure of an enzyme in solution is generally very
similar to its structure in the solid state, i.e. the crystal
Importance of knowing 3-D structure of an
enzyme
To test models of macromolecular structure
• Success of structure prediction methods can be tested
To propose a mechanism of catalysis
• Structural data can be combined with other studies (e.g kinetics) and
chemically reasonable mechanisms can be proposed
To explore similarities btw enzymes
• Add more to a.acid comparison
– e.g. common 2o structural elements in a number of nuclotide binding
enzymes
To assist rational drug design
• With powerful computational methods, new ligands can be designed
Subunits and Quaternary Structure-1
• In many cases, the biological activity of a protein requires the
presence of more than one polypeptide chain
• Individual polypeptides are referred as subunits
– Subunits have their own secondary and tertiary structures
– They are held together by non-covalent interactions and may additionaly
have covalent disulfide bonds
• The arrangement of subunits defines the quaternary structure
• They may have different arrangements existing in equilibrium with
each other
• Arrangement can greatly affect the function:
– e.g. In hemoglobin, affinity of heme groups for oxygen is affected and
leads to the reversibility of oxygen binding...
Subunits and Quaternary Structure-2
Significance of subunits:
1. Additional possibilities of regulating the catalytic activity
2. Variation in catalytic properties: e.g. RNA polymerase or lactose
synthase ( and  subunit)
3. Increased stability of active configuration
4. Association can generate large structures of defined geometry
which may be required for particular function (e.g. Chaperonin 60,
14 identical subunits...)
Subunits and Quaternary Structure-2
Folding – Unfolding
• Compact folded form is generally thermodynamically more stable
BUT by only a relatively small margin (20-60 kj/mol)
 Most enzymes can be easily unfolded by changing medium conditions
• Folded  molten globule  unfolded
• Early 60s, Anfinsen  Primary structure dictates 3-D structure
• Current view  formation of small local elements  arrangement to
bury the hydrophobic side chains  elements of 2O str. act as
nucleation centers to direct further folding  3O interactions are
formed
• Refolding of proteins with multiple domains or subunits is much less
efficient
Subunits and Quaternary Structure-3
For proper folding
• Co-translational folding
• 1980s  chaperones
• Protein disulphide isomerase and peptidyl prolyl isomerase
Subunits and Quaternary Structure-4
Structure prediction methods
• Homology modelling: > 25 % sequence identity to one of known 3-D
structure
• Calculate most stable conformation
– Laborious and useful for very short peptides
• Semi-empirical approaches
– Use 3-D structure databases as a guide
e.g. Glutamic acid show preference for -helices but proline not
e.g. Use of neural network methods + multiple sequence alignments
World wide web
• European Bioinformatics Institute (EBI)-Enzyme structures
database
http://www.ebi.ac.uk/thornton-srv/databases/enzymes/
• ExPASy Proteomics Server and SWISS-PROT database
• Protein Data Bank