Introduction to Protein and Enzyme Biochemistry Lecture Slides PDF

PH1123 Introduction to Protein and Enzyme Biochemistry Three Lectures Dr Alex White [email protected]  What are Proteins? PH1123 Proteins discovered 1838. Proteios: first, primary. These molecules were very abundant in biological systems; they were clearly very...

PH1123 Introduction to Protein and Enzyme Biochemistry Three Lectures Dr Alex White [email protected]  What are Proteins? PH1123 Proteins discovered 1838. Proteios: first, primary. These molecules were very abundant in biological systems; they were clearly very important, but their precise function was unknown. Subsequently determined that proteins were polymers. Nature uses 20 distinct monomers (amino acids) to build all proteins. Amino acid general formula Amino acids fall into seven distinct classes based on the chemical properties of the sidechain, (R): [Aliphatic, Aromatic, Alcoholic, Sulfur Containing, Acidic, Basic or an Amide] The amino acid sidechain provides a specific chemical property to the protein. Each amino acid is identified by a three letter code or a one letter code.  What are Proteins? PH1123 Bonding between carboxylic acid and amino groups of two adjacent amino acids forms a continuous peptide backbone with protruding R groups: The continuous peptide backbone folds into a specific three dimensional shape. Every protein has a unique and complex structure. This is easier to visualise by studying the backbone alone: The same enzyme in spacefill shows all atoms (except hydrogen). It is harder to visualise features, but demonstrates the compact, spherical nature of enzymes.  Biological Functions of Proteins PH1123 The complex structures of proteins allow them to undertake a wide variety of functions: Catalysis Proteins that catalyze chemical reactions in the body are known as enzymes. Several thousand known. Storage and transport Ferritin stores and transports iron in the body. Haemoglobin transports oxygen. Mechanical support and shape Collagen is a component of supportive tissue e.g. ligament, skin, bone etc. Most abundant protein found in vertebrates. Decoding information, gene expression RNA polymerase synthesizes RNA (directed by DNA). Specialist Functions Immunoglobins (antibodies) and some hormones (e.g. insulin) are proteins. Almost every function in the human organism is in some way controlled by a protein  Aliphatic Amino Acids: Contain Alkyl Sidechains PH1123 The aliphatic amino acids include the simplest amino acid, glycine, R= H Valine, Val, V Glycine, Gly, G Alanine, Ala, A Leucine, Leu, L Isoleucine, Ile, I Proline, Pro, P The alkyl side chains contain no functional groups, but are very hydrophobic [water hating].  Aromatic Amino Acids: Contain an Aromatic Ring PH1123 Tryptophan, Trp, W Phenylalanine, Phe, F Tyrosine, Tyr, Y The aromatic amino acids Phe, Trp and Tyr are quite hydrophobic. Alcohol Containing Amino Acids Serine, Ser, S Threonine, Thr, T Ser and Thr have aliphatic side chains containing a hydroxyl group. As a result they are hydrophilic [water loving] since they can interact (via hydrogen bonding) with water and other polar groups. They contain a function group (an alcohol) and are chemically reactive.  Sulfur Containing Amino Acids PH1123 Cysteine, Cys, C Methionine, Met, M The sulfur containing amino acids are hydrophobic in character. The -SH (thiol) group in cysteine is very chemically reactive. Acidic Amino Acids Aspartic Acid, Asp, D Glutamic Acid, Glu, E Asp and Glu are acidic and very hydrophilic. Also, they have chemical reactivity and are often found in the active sites of enzymes.  Basic (nitrogen containing) Amino Acids PH1123 Histidine, His, H Lysine, Lys, K Arginine, Arg, R Lys, Arg and His are basic, they have polar sides so they are very hydrophilic. They are often found in the active sites of enzymes. Amide Containing Amino Acids Amides are neutral and chemically less reactive than carboxylic acids. They Glutamine, are still very polar and Gln, Q therefore hydrophilic. Asparagine, Asn, N  Essential and non-essential amino acids PH1123 Some amino acids are only biosynthesized by plants or microorganisms. These must be obtained from the diet & are known as essential amino acids. Essential Non-Essential Arginine, (Arg) Alanine (Ala) Histidine (His) Asparagine (Asn) Isoleucine (Ile) Aspartic Acid (Asp) Leucine (Leu) Cysteine (Cys) Lysine (Lys) Glutamic Acid (Glu) Methionine (Met) Glutamine (Gln) Phenylalanine (Phe) Glycine (Gly) Threonine (Thr) Proline (Pro) Tryptophan (Trp) Serine (Ser) Valine (Val) Tyrosine (Tyr) Non essential amino acids can be biosynthesized within the body if required. However, they are mostly obtained from the diet.  Other Amino Acids PH1123 3-Methyl histidine, -Carboxyglutamate found in found in blood clotting proteins histones Other amino acids, in addition to the common 20 do exist. However, they are generally very rare and are not coded by DNA (they are biosynthesized). Tyr Dopamine His Histamine Amino acids are also used as starting materials for the biosynthesis of important molecules within the body. The neurotransmitters Dopamine and Histamine, involved in shock and allergic response, are derived from amino acids.  Acidity / Basicity of Amino Acids PH1123 The human body is mostly aqueous (approx. 80 % water) and buffered at pH 7.4 (physiological pH). Under these conditions, free amino acids are dipolar and exist as ‘Zwitterions’. For an amino acid in solution the ionisation state varies with pH. Acidic, low pH Basic, high pH The side chain functional groups are also effected by local pH conditions. At physiological pH, acidic side chains are deprotonated and Lysine basic side chains are protonated. Aspartic acid (aspartate)  Stereochemistry of Amino Acids PH1123 Any carbon atom with 4 different substituents is asymmetric or chiral: Molecules containing a chiral centre are able to rotate plane polarized light in a clockwise (+) or anti- clockwise (-) direction: they are optically active. A molecule with a chiral carbon can exist as enantiomers: compounds with the same molecular formula but differing in their configuration in space Consider an amino acids mirror image: 180º They are two different isomers (enantiomers) and are non-superimposable  Stereochemistry of Amino Acids (2) PH1123 To describe different enantiomers, pairs of amino acids are designated D or L. The reference compound is glyceraldehyde (a sugar). The lowest priority group The most highly (see Stereo chemistry oxidised group next lectures) can be placed to the chiral centre is to the right or left placed at the top L-Glyceraldehyde D-Glyceraldehyde L-Serine D-Serine L-amino acids have the same configuration at their chiral carbon as L-glyceraldehyde All the naturally occurring amino acids have the L-configuration, except glycine. Some organisms synthesize D-amino acids. [The D and L system is arbitrary and should not be confused with dexotrorotatory (+) and levororotatory (-). L-amino acids can rotate plane polarized light in either directions. e.g. L-proline (-86.2°) and L-arginine (+12.5°)]  Protein Synthesis: Nature PH1123 The central dogma: DNA  RNA  Proteins DNA mRNA enzyme strand ribosome A Protein See Nucleic Acids lectures! Also Try: www.youtube.c tRNA om/watch? v=gG7uCskUOr A  Protein Synthesis: Laboratory PH1123 A condensation reaction between the amino group and carboxyl group of two amino acids results in formation of a Peptide Bond: Ala Ser Peptide Bond A dipeptide [Ala-Ser] has been formed. A peptide is a small protein of less than 50 amino acids. This reaction can be repeated to synthesize a tripeptide: Ala-Ser-Phe + N Terminal C Terminal By convention, peptides should be named / drawn from the amino (N) terminal on the left to the carboxy (C) terminal on the right: e.g H2N-Ala-Ser-Phe-CO2H  Chemical Steps in Laboratory Peptide Synthesis PH1123 Protection. Blocking of the carboxyl and amino groups not involved in the required peptide bond. Activation of the carboxyl group involved in peptide bond formation. Hydroxide is a poor leaving group The carboxyl group can be activated by conversion to an acid chloride. Chloride (Cl-) is a better leaving group Coupling. Reaction of the free amino group of the second amino acid with the activated carboxyl group to form a new peptide bond. Deprotection. Removal of the protecting groups.  Chemical Steps in Peptide Synthesis (2) PH1123 Amino protection Carboxy protection P1= Protecting Group P2= Protecting Group + Activation Loss of HCl Deprotection Ala-Gly  Uses of Peptides PH1123 H2N-Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu- Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu- Phe-Lys-Asn-Ala-Ile-Val-Lys-Asn-Ala- His-Lys-Lys-Gly-Gln-COOH H2N-Asp-Phe-CO2Me Aspartame, synthetic dipeptide -Endorphin, an endogenous 31 amino 200 sweeter than sugar acid neuropeptide with analgesic effects. Arg-Gly-Asp-Trp-Pro Eptifibatide (Integrilin ®) ….is a cyclic, pseudo-peptide (i.e. contains cardiovascular antiplatelet drug…. amino acids and a non peptide region).  Uses of Peptides 2 PH1123 Ciclosporin is a widely used immunosuppressant drug, isolated from natural sources, that prevents rejection following organ and tissue transplantation. It is a cyclic peptide that contains naturally occurring and modified (microorganism biosynthesis) amino acids: Naturally occurring amino acid: Alanine Slightly modified glycine; an N-methyl group has been added Non-naturally occurring amino acid with unusual side chain Peptides can be very useful therapeutic molecules but are also very readily metabolised (broken down) particularly in the stomach.