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Enzymes II Coenzymes, Regulation, Catalytic Antibodies and Ribozymes Enzyme:Coenzyme Partners Allosteric Enzymes Cellular Regulation of Enzymes Site-Directed Mutagenesis and Catalytic Antibodies Catalytic RNA 1 Enzyme:Coenzyme Partners Coenzyme Organic or organometallic molecule that assists an enzyme. Prosthetic group Coenzymes that are covalently linked or noncovalently bound very tightly to an enzyme partner. Vitamins A group of relatively small, organic molecules essential for proper growth and development. 2 Cofactors and coenzymes Some enzymes require a second species to be present in order to do their job. For Cofactor type enzymes: Apoenzyme - protein portion of enzyme - almost ready to work. Cofactor - prosthetic group needed to ‘activate’ the apoenzyme. - usually a metal ion that holds protein in the proper shape. 3 Cofactor example Co2+ non reacting apoenzyme Co2+ 4 Coenzymes A second species that temporarily binds to the apoenzyme in order for it to work. + apoenzyme coenzyme holoenzyme 5 Vitamins Thirteen well-identified vitamins that are classified by their water solubility. Part of each coenzyme structure is made from a vitamin. Coenzyme NAD+ FAD Coenzyme A Vitamin required niacin riboflavin pantothenic acid 6 Water-soluble vitamins Pantothenic acid One of the B vitamins. Abundant in many foods. It is also manufactured by intestinal bacteria. HO O OH CH3 || | C - CH2 - CH2 - N - C - C - C - CH2OH | | | H H CH3 | O Uses Undefined role in metabolism of proteins fats and carbohydrates. Believed to be converted to coenzyme A. 7 Water-soluble vitamins Niacin - Vitamin B3 Refers to both nicotinic acid and nicotinamide. Found in fish, meat, milk, cereals. O O || || -C-OH -C-NH2 Uses N nicotinic acid N nicotinamide Works as a coenzyme in the release of energy from nutrients. Precursor to NAD+ and NADP+. 8 Water-soluble vitamins Riboflavin - Vitamin B2 Found in milk, eggs, leafy green vegetables. N N-H N N | CH2-CH-CH-CH-CH2 | | | Component of H3Cthe coenzymeH C3 FAD. Plays a role in the metabolism of carbohydrates, fats and respiratory proteins. O || | | Uses H OH OH OH OH 9 Water-soluble vitamins Thiamine - Vitamin B1 Found in meat, cereals, leafy green veggies. CH3 | NH2 H C=C-CH2CH2OH | | + N - C-N | C-S H | H3CH N Uses Required for many decarboxylation reactions. Catalyst in carbohydrate metabolism. Enables pyruvic acid to be absorbed and carbohydrates to be released. Also plays a role in the synthesis of nerve-regulating substances. 10 Water-soluble vitamins Pyridoxine - Vitamin B6 Found in fish, meat, poultry, green leafy vegetables. R | -CH2-OH HOH3C- If R = -CH2OH, If R = -CHO, If R = -CH2NH2, Pyridoxine Pyridoxal Pyridoxamine N Uses. Plays role in the synthesis of red blood cells and the use of fats. Required in synthesis and breakdown of amino acids. 11 Water-soluble vitamins Folic acid - A B vitamin. Found in meat, cereals, leafy green vegetables, intestinal bacteria. Stored in liver. OH N H2 C N O COOH C NH N H H2N Uses N N CH2 CH2 COOH Synthesis of purines and pyrimidines. Coenzyme needed for forming body protein and hemoglobin. 12 Water-soluble vitamins Biotin - A B vitamin. Found in liver, egg yolks, cheese, peanuts. Synthesized by intestinal bacteria. H2 C S H2 C C H2 COOH C H2 Uses N N H H O Involved in carboxylation and decarboxylation in metabolism of fats, carbohydrates, and proteins. 13 Water-soluble vitamins H2NC=O Uses Production of red & white blood cells. Growth & maintenance of nerve tissue. H2NCH2C H3C H3C || H2NCCH2 O=C-CH2CH2 NH HC HO-CH2 CH3 CH3 N N CH2 C O Co+ O CH2 CH-CH3 O OP O O CH2 CN N N N Found in meat, eggs, dairy products. Most recently discovered vitamin. || CH2CNH2 || N Vitamin B12 - Cobalamin O CH2 CH3 CH3 O NH2 CH3 CH3 CH2 CH2 C O CH3 CH3 NH2 OH C C H H O C H 14 Water-soluble vitamins Vitamin C - ascorbic acid Found in fresh fruit and vegetables. OH O Uses HO O CH-CH2OH OH Formation and maintenance of collagen. Enhances absorption of iron from foods. Serves as an antioxidant. 15 Lipid-soluble vitamins Vitamin A - trans-retinol Found in liver, egg yolks, green and yellow leafy vegetables, fruit. H3C Uses CH3 | -(CH=CH-CH=CH)2-CH2-OH CH3 CH3 Maintains skin and mucous membranes of oral cavity, and digestive, respiratory, reproductive, and urinary tract. Critical for vision. 16 Lipid-soluble vitamins Vitamin K1 - phylloquinone Found in leafy vegetables and intestinal bacteria. O CH3 H C C H2 CH3 C CH 3 (CH2CH2 C CH2)2 CH 2 H CH 3 CH2 C H CH 3 O Uses Essential in blood clotting. Aids in the formation of prothrombin - the enzyme need to produce fibrin. 17 Lipid-soluble vitamins Vitamin D - cholecalciferol Found in liver and fish oils. Produced in body when exposed to light. H3C H3C Uses CH3 CH3 CH2 Necessary for normal HO bone formation and for retention of calcium and phosphorus. 18 Lipid-soluble vitamins Vitamin E - tocopherol Found in vegetable oils, wheat germ, liver and green leafy vegetables. H3C Uses O H3C OH | CH3 H3C H H3C H CH3 H3C Role of this vitamin is not clearly established. CH3 19 Coenzymes Let’s look at a few coenzymes that are produced from enzymes. NAD+ nicotinamide adenine dinucleotide. FAD flavin adenine dinucleotide. Coenzyme A 20 NAD+ reactive site O O O C - P O CH2 O O O O CH2 - ribose nicotinamide N+ O OH P NH2 OH N N O OH NH2 N adenine N OH 21 FAD O H3C N H3C active site is highlighted NH N N H C H H C OH H C OH H C OH H C H O riboflavin NH2 N O O P O O - ribose CH2 N O OH N adenine N OH 22 Coenzyme A phosphorylated ADP pantothenate unit NH2 O O H CH3 C-CH2-CH2-N-C-C-C-CH2 H HO CH3 H-N O O N N P O P O O- O- CH 2 O N N CH2-CH2 S H Sulfhydryl group O OH O P OO23 Allosteric enzymes The thousands of reactions involved in metabolism are grouped in sequences. A E1 B E2 C E3 D E4 F E5 P While the behavior of most of the enzymes can be explained by Michaelis-Menten kinetics, there is typically a step that does not. This step involves a regulatory enzyme that controls the rate for the entire sequence. 24 Allosteric enzymes A number of factors can influence a regulatory enzyme. • concentration of the final product(s) • beginning substrate sequence • intermediates formed in the pathway • an external factor like a hormone • a combination of the above 25 Allosteric enzymes In many pathways, the first enzyme is often the major control enzyme. A E1 B E2 C E3 D E4 F E5 P • It is influenced by the concentration of the starting material • It may also be affected by the amount of final product. 26 Methods of enzyme regulation End product inhibition Enzyme - substrate reaction is an equilibrium If product builds up, the reaction slows. E+S ES ES* EP E+P Equilibrium shifts to left if product starts to build up 27 Positive and Negative Effectors Effectors or modulators These are molecules that serve to alter how an enzyme performs. Positive effectors stimulate an enzyme Negative effectors inhibit an enzyme They act by reversible, noncovalent binding to a site on the enzyme. This alters the conformation of the active site. 28 Methods of enzyme regulation Use of allosteric enzyme Similar to coenzymes. Example of positive allosterism. 29 Cellular regulation of enzymes Covalent modification Reversible, covalent changes to specific amino acid side chains. Common alterations • Phosphorylation of hydroxyl groups in serine, threonine or tyrosine. • Attachment of an adenosyl monophosphate (AMP) to a similar hydroxyl group. • Reduction of cysteine disulfide bonds. 30 Covalent modification Enzymes are used to convert the regulatory enzyme to either an active or inactive form. Example - control of glycogen phosphorylase phosphorylase + 2 ATP phosphorylase + 2 ADP OH OP OH OP Specific serine residues in each of two identical dimers of the enzyme are phosphorylated. The reaction is catalyzed by phosphorylase kinase. The process can be reversed using a second enzyme, phosphorylase phosphatase which effects the removal of phosphate. phosphorylase + 2 H2O phosphorylase + 2 Pi OP OH OP OH 31 Covalent modification Other examples Attachment of AMP to glutamine synthetase enzyme + ATP OH active form enzyme + PPi O-AMP inactive form Reduction of cysteine disulfide bonds by AH2 enzyme + AH2 enzyme + A S S SH SH active form inactive form 32 Cellular regulation of enzymes Activation of proteolytic cleavage Some enzymes are initially produced in an inactive form - zymogen. A portion of the protein chain must be removed to make it active - proteolytic cleavage. This is irreversible. inactive form zymogen active form 33 Chymotrypsin This enzyme is a proteolytic enzyme. produced from chymotrypsinogen. Initially, -chymotrypsin is formed is and subsequently converted to -chymotrypsin. In its final form, it consists of three polypeptides held together by two interchain disulfide bonds. This enzyme cleaves peptide bonds. It only works on amino acids containing an aromatic ring phenylalanine, tyrosine & tryptophan. 34 Chymotrypsin 1 245 chymotrypsinogen (inactive) trypsin 1 15 16 245 Arg Ile chymotrypsin 1 13 16 Leu Ile -chymotrypsin (active) Ser - Arg and Thr - Asn 14 15 147 148 146 Tyr 149 Ala 245 -chymotrypsin (active) 35 Another example Blood Clotting - formation of fibrin. Process requires a series of enzymatic steps. Many of the enzymes are made as inactive forms to reduce clotting on its own. Two pathways can be used to start the process. Extrinsic - Activated by tissue damage, outside the blood vessel. Intrinsic - Activated by damage within a blood vessel. 36 Summary of pathways Extrinsic pathway Activation Intrinsic pathway Activation XII VII XII* XI VII complex* XI* IX VII* IX* VII complex* Common pathway X X* prothrombin fibrin thrombin fibrogen fibrin polmer 37 Fibrin Ribbon model of fibrin. 38 Drug interactions Drugs can be administered to alter the clotting mechanism. Example: Heparin - an anticoagulant. Acts by accelerating the action of the existing inhibitor of thrombin - antithrombin III. Antithrombin III inhibits activating the clotting factors that have a reactive serine residue at their enzymatically active centers. 39 Heparin interaction thrombin antithrombin 40 Heparin interaction thrombin serine antithrombin inhibited thrombin lysine sites heparin Addition of heparin makes it easier for thrombin to interact with antithrombin - positive allosteric effect. 41 Cellular regulation of enzymes Isoenzymes or isozymes • Enzymes that have similar but not identical amino acid sequences. • Each will catalyze the same biochemical reaction. • They differ in kinetics - different KM and Vmax values. • Use different effectors and forms of coenzymes. • Cellular distribution of each form will vary. 42 Regulation by isoenzymes The best know example is lactate dehydrogenase (LDH) which catalyzes the conversion of pyruvate to lactate in muscle tissue. It is a tetramer composed of two possible types of subunits - M and H These units are made by separate genes and have similar but different amino acid sequences. 43 LDH isoenzyme In skeletal muscle, the predominate form is M4. This varies for other tissues. In the heart, the form is H4. In the liver, every possible combination is observed: M4, M3H, M2H2, MH3 and H4. The reasons for isoenzymes are not well understood but may allow regulation based on different metabolic patterns. 44 LDH isoenzyme - M4 45 Site-directed mutagenesis and catalytic antibodies Studies have shown that catalysts for biochemical reactions are not limited to naturally occurring proteins. Site-directed mutagenesis Modification of amino acid sequences of known enzymes and other proteins. Catalytic antibodies Production of protein antibodies using transition-state analogs 46 Site-directed mutagenesis Using recombinant DNA procedures, it is possible to modify a gene to use a different amino acid in a protein sequence. • Assists in the study of enzyme structure and activity. • Allow for the design of new enzymes and other proteins with desired properties. • The approach can be used for the design of new drug therapies. 47 Catalytic antibodies Protein antibodies function by tightly binding and neutralizing foreign substances (antigens). Pauling proposed that antibodies were similar to enzymes but bound substrate molecules in the transition state. Studies have been conducted to see if an antibody with enzyme-like activity could be produced. 48 Catalytic antibodies An example. Hydrolysis of the methyl ester of p-nitrobenzoate. O O2N C O- H2O OCH3 O2N C OCH3 H O+ H transition state The goal was to produce an antibody that bound the proposed tetrahedral transition state which is very unstable. 49 Catalytic antibodies An analog that was easily prepared was studied. O O2N P CH 2CH 2CH2CH 2COO- O- It was directly injected into animals and found to result in antibody production. This antibody catalyzed reaction was found to obey Michaelis-Menten kinetics and accelerate rate on the order of 103-106 fold. 50 Catalytic RNA Since their discovery, it was believed that all enzymes were proteins. In 1981 - 1982, two research group reported results on catalytic RNA. In 1989, the Nobel Prize in Chemistry was awarded to Sidney Altman (Yale) and Thomas Cech (University of Colorado Boulder) for their discovery. The term ribozyme is now used for RNA enzymes. 51 Ribonuclease P This was the first type of catalytic RNA discovered and is present in all organisms • Substrates are at least 60 inactive, precursor forms of tRNA. • Ribonuclease P acts to remove a segment of the ribonucleotide, producing mature tRNA. • The enzyme consists of a small protein subunit with a molecular weight of 14,000 and an RNA component of 377 nucleotides. 52 A A G A C ppp G Ribonuclease P U C G C G U G U A C G H O- C A C A G G C C A G C A U U A C C C C Portion removed by RNase P RNase P functions by hydrolyitc cleavage of the phosphodiester bond. G A U G C U C G G G U G C C G A The enzyme obeys Michaelis-Menten kinetics, is only needed in small amounts and must remain in its native tertiary structure for activity. G G U G C U A G C G C G U C G G C U U G C A G G C C U C C G G U U A G C C C G C U G C G U A C G C G C G U A G G U G C G C 53 Self-splicing RNA introns The second example of catalytic RNA reported involved studies of intron splicing in Tetrahymena thermophila. It was observed that for this protozoan, splicing of an intron in rRNA was autocatalytic - it cleaved itself. This splicing cut out an intron sequence of 414 nucleotides which was later processed to L-19 IVS RNA, lacking 19 nucleotides. 54 Self-splicing RNA introns 5’ upstream exon 5’ G OH .. G OH downstream exon 3’ 3’ 55 Self-splicing RNA introns 5’ 5’ - 19 nucleotides Spliced exons L-19 IVS RNA + 414 nucleotides 3’ 3’ 56 Self-splicing RNA introns List of activities for catalytic RNA • Cleavage and rejoining of oligonucleotide substrates. • Cleavage of DNA phosphodiester bonds. • Cleavage of RNA at sequence-specific sites. • Hydrolysis of esters. • Formation of peptide bonds between amino acids. 57 Significance of ribozymes Other forms of catalytic RNA continue to be discovered. • Small ribozymes have been found as components of plant RNA viruses. • The active region of this RNA consists of only 19 - 30 nucleotides. • Because of their characteristic shape and action, they are called “hammerhead” ribozymes. 58 Hammerhead ribozyme 59