lecture_3 - Classes of Proteins Enzymes - The largest class...

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Unformatted text preview: Classes of Proteins Enzymes - The largest class of proteins. These proteins are catalysts. They accelerate the rates of the various biological reactions that take place in the cell. Almost every reaction that occurs in biochemistry is facilitated by some sort of enzyme. They are typically named based on the reaction that they catalyze, and have suffixes with the letters -ase. Examples include proteases (trypsin, chymotrypsin, carboxypeptidases), lactase (hydrolyzes milk sugar), Regulatory Proteins - Don't perform any chemical transformation. They regulate cellular processes based on their abilities to bind to other macromolecules, such as receptors, DNA, RNA, etc. A well known example of course is insulin, the hormone that regulates glucose metabolism. Insulin is a fairly small protein (5.7 kD), and is composed of two identical polypeptide chains that are linked via a disulfide bond. Transport Proteins - Well known example is hemoglobin. Many others are membrane bound proteins which aid in shuttling nutrients or metabolites (glucose, vitamins, amino acids) across the cell membrane. Storage Proteins - Just polypeptides of amino acids that can be broken down to the individual amino acid in order to extract nitrogen. Nitrogen is frequently a limiting nutrient for growth. Proteins can also store metal ions. Metal ions, which are positively charged, can bind to negatively charged side chains, or side chains that are very polar. Ferritin, is the common animal storage protein for iron. Reading, Chap. 3 (57-63), Chap 5 (115-126). Classes of Proteins II Contractile and Motile Proteins - Endow cells with the capacity for special forms of movement. Actin and Myosin (muscle fibers). Tubulin, major protein component of microtubules, which facilitate cell division. Structural Proteins - Provide strength and protection to cells. They are typically insoluble. akeratins are the major proteins of the skin, hair, and fingernails. Collagen is the major protein in bone, connective tissue, tendons, and cartilage. Silk is also a polymer of a structural protein, fibroin, which is a b-keratin. Scaffold Proteins - These proteins act as adaptors. They bind to other proteins, and form a scaffold upon which certain protein complexes or protein-DNA complexes e.g. can be built. They can also help to mediate certain reactions between two proteins. Protective and Exploitive Proteins - Immunoglobulins or antibodies, which are made by lymphocytes, and function in the immune system by locating and neutralizing molecules that are not intrinsic to the host cell. Thrombin and fibrinogen, which are blood clotting proteins, which help to prevent severe loss of blood upon injury. Antifreeze proteins help to prevent the blood of arctic and antarctic fish from freezing. Also include toxins, like those from poisonous snakes, or even bacteria. Amino Acid Structure From Garret and Grisham (2nd Ed) Amino acids are the building blocks of peptides and proteins, the majority of which are composed of 20 commonly occurring ones. The properties of amino acids that make them suitable for the varied roles of proteins is that 1. They have the capacity to polymerize 2. They have novel-acid base properties 3. They have varied structure and chemical functionality 4. They are Chiral Amino acids are composed of a tetrahedral arrangement of 4 substituents around a central carbon atom, called the alpha carbon. All (except proline) have a common carboxyl group (the acid), amino group, and an alpha hydrogen. They differ only in their R substituent. The Peptide Bond What allows amino acids to polymerize to form peptides and proteins is the unique covalent linkage called a peptide bond. The bond is the result of a head to tail condensation of the amino group of one amino acid and the carboxyl group of another. Formation of this bond results in the release of 1 mol of water per mol of peptide bond formed. This reaction does not happen without the input of energy in the form of ATP. This is because the equilibrium in aqueous solution lies far in favor of hydrolysis. In the cell (with a few exceptions), peptide bond formation in proteins takes place on the ribosome. Nonpolar Amino Acids Amino acids can be placed in four basic groups depending on their R substituents. All Amino acids have a three letter code and a one letter code for shorthand notation. You are responsible for knowing them as well as the structures of each amino acid. Proline should also be on this slide! It is also a nonpolar amino acid. Nonpolar, Aromatic Amino Acids Polar, Uncharged Amino Acids These amino acids are important in hydrogen bonding and disulfide bond formation Proline is a nonpolar amino acid. The mistake was corrected in your Textbook, but not on the figures CD. Another related amino acid is selenocysteine, in which the sulfur in cysteine is replaced by selenium. This amino acid is incorporated in proteins via ribosomal processes similar to the standard 20 amino acids. Notice that the R group is covalently bonded to the a-amino group Postively Charged (Basic) Amino Acids These amino acids frequently form ionic interactions as well as hydrogen bonds. They are positively charged at neutral pH. Negatively Charged (Acidic) Amino Acids These amino acids can form ionic interactions and hydrogen bonds. They are negatively charged at neutral pH. Memorize Rationalize Less Common Amino Acids Here are some amino acids that are found in proteins, but are comparatively rare. They are not synthesized by ribosomal processes; most typically arise from post-translational modifications to the protein, which are catalyzed by specific enzymes. Common post-translational modifications include hydroxylation, methylation, acetylation, and phosphorylation. You are not responsible for knowing these amino acids, however, if asked you should be able to recognize that they are not one of the common 20. Chirality of Amino Acids All amino acids have four different substituents around the alpha carbon. There is one exception. This configuration is asymmetric, and carbons with four different substituents are called chiral carbons. Remember that configuration is distinct from conformation. A configuration can be changed only by breaking covalent bonds. The two different configurations about a chiral carbon are called enantiomers. They are non-superimposable mirror images. Enantiomers display optical activity. That is, they can rotate the plane of polarized light. Clockwise rotation is called Dextrarotatory, while counter-clockwise rotation is called Levorotatory. The magnitude and direction will depend on the specific enantiomeric substance. In the case of amino acids, it will depend on the R group. Notice how the two stereoisomers of glyceraldehyde are mirror images but nonsuperimposable. Systems of Nomenclature The two systems that are in common use to denote the configuration of chiral centers are the D,L system, and the R,S system. In practice, the D,L system is used mainly for amino acids and simple sugars, whereas the R,S system is used for most other organic compounds. The D,L system is based on the configuration of glyceraldehyde. All standard amino acids are of the L configuration, and the arrangment of constituents about the alpha carbon are similar to that of Lglyceraldehyde. Notice that the structures on the left are Fischer projections. Solid lines indicate that the atoms are coming out of the plane of the board, while the dashed lines indicate that the atoms are going into the plane of the board. The R,S System of Nomenclature Notice that some amino acids have more than one chiral center. These include isoleucine, threonine, as well as the less common amino acids hydroxyproline and hydroxylysine. Isomers that differ in configuration at only one of the asymmetric centers are called diastereomers. The R,S system was developed as a better method for dealing with multiple chiral carbons. In the R,S, system, atoms are given priority based on their atomic number. SH>OH>NH2>COOH>CHO>CH2OH>CH3 View the molecule in such a way that the atom with least priority is facing away from you. With the remaining atoms, count backwards from highest to lowest configuration. If you must count counterclockwise, the configuration is S. Clockwise is R. Ionization of Amino Acids The ionization of the alpha amino and alpha carboxyl groups of amino acids will allow three distinct states. At neutral pH, the Zwitterionic state predominates. In this state, the overall charge (disregarding the R group) is 0. At significantly higher pH, loss of a proton from the amino group occurs. This gives the anionic (-1 charged) form. Notice how the pKas for the carboxyl ionization differ from that of acetic acid in solution. You should be familiar with the pKa values of ionizable side chains. I won't ask you to repeat any pKa values, but I may ask what is the net charge on a particular amino acid is at a given pH. Amino Acids Can Act as Buffers pI Because amino acids are weak acids, they can act as buffers. Notice that glycine can act as a buffer in two pH regimes. The pKa of its carboxyl group is 2.3, while that of its amino group is 9.6. The average of the two pKas represents the Isoelectric Point (equivalence point, pI)of the amino acid. This is where it is fully Zwitterionic (carries a net charge of 0), and where it is least soluble. pI = 1/2(pK1 + pK2) What is the pH of a glycine solution in which the a-NH3+ group is one-third dissociated? Titration Curves and Isoelectric Point Some amino acids can be identified by their titration curves. Notice how the isoelectric point (pI) is determined for both of these amino acids. The Ninhydrin Reaction H2O The Amino Acid Cysteine Important biological reactions in which cysteine is involved. Disulfide bond formation Covalent modification Cysteine quantification Spectroscopic Detection of Amino Acids The aromatic amino acids absorb light in the ultraviolet region, giving rise to characteristic spectra. Proteins typically have UV absorbance maxima around 275-280 nm, which is almost entirely due to the absorbance properties of tryptophan and tyrosine. Notice that the ordinate is a log scale. Tryptophans molar absorptivity is 5 times that of tyrosine, and 50 times that of phenylalanine. By knowing the Molar absorptivity of a particular protein, its concentration can be readily determined by UV-visible spectrophotometry using Beers law (A = ebc). Molar Absorptivity Many biological molecules absorb UV-visible light, and measurement of light absorption can be done routinely using a spectrophotometer. A monochromator is used to allow only a specific wavelength of white light to be shown on a sample at one time. The intensity of the light that hits the detector I (i.e. not absorbed) is measured as a function of wavelength and intensity of light that is shown on the sample Io. The absorbance is governed by the equation log(Io/I) = ecl, where c = concentration (M), l is the path length of the sample (cm), and e is the molar absorptivity of the sample (M-1 cm-1). The molar absortivity varies with the nature of the absorbing compound, and is wavelength dependent. This is referred to as the Lambert-Beer law, or just Beer's law. How Many Cysteines? An unknown peptide was determined to have a Molar absorptivity at 280 nm of 10,000 M-1cm-1. A 1 mL solution of the peptide (1 cm cuvet) has an absorbance at 280 nm of 0.5. Upon addition of DTNB to the 1 mL solution of peptide, the solution turns dark yellow. The absorbance of the solution at 412 nm (1 cm cuvet) is 1.14. Calculate the number of cysteine residues in this peptide. Separation and Analysis of Amino Acid Mixtures The ability to separate amino acids in mixtures is usually based on relative differences in their physical and chemical traits. These are all mediated by their "R" or functional groups. The general strategy is to exploit the ability of a given amino acid to partition between two different phases. It could be two liquid phases (extraction), a solid-liquid phase, or a gas-liquid phase. In particular, solid-liquid phase methods are routinely used, the procedure being termed chromatography. Typically, the first strategy is to exploit any charge differences among the amino acids at a given pH. This is done by ion-exchange chromatography. If you were given a mixture of amino acids, K, A, G, R, E, and H, could you predict their relative elution pattern from a cation exchange column at pH 7.0? Dp = pI pH In what order would this molecules elute on a Cation-Exchange Column at pH 6? Amino Acid Separation Unfortunately, amino acids are not colored as described in this overhead. Therefore, what methods would you use to first check if an amino acid is indeed present? ...
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This note was uploaded on 10/18/2008 for the course BIO 120 taught by Professor Ashena during the Spring '06 term at Punjab Engineering College.

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