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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