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LECTURE 2: ENZYME Ribonuclease structure showing histidine probes Enzymes are critical for every aspect of cellular life What is an important enzyme that you are familiar with? PAPAIN What is papain? Papain is a protein-cleaving enzyme derived from papaya and certain other plants. Enzymes are complex molecules produced in living organisms to catalyze (speed up) chemical reactions within the cell PAPAIN Why is meat tough? Muscles have to endure a lot of mechanical stress; they are made of strong fibers that make them hard to cut, and tough connective tissue holds them together. Individual muscle cells contain microscopic fibrils that give them their structural integrity and allow them to contract. The fibrils have a complex internal structure bound together by long protein chains. The connective tissue that holds the muscle together is also mostly protein. How does papain tenderize meat? Papain cuts the protein chains in the fibrils and also in the connective tissue, disrupting the structural integrity of the muscle fiber, and tenderizing the meat. 1. DEFINITION ENZYMES ARE METABLOLIC CATALYSTS. Enzymes (in yeast) are the largest and most highly specialized catalysts in the body for the reactions involved in metabolism which increase the rate of chemical reactions by lowering the activation energy of that reactions ENZYME TOPICS 1. 2. 3. 4. 5. Functional Properties Nomenclature Enzyme Specificity Enzyme Regulation Activation Energy 1. Enzyme Properties ENZYMES ARE 1. Proteins (note that recent developments indicate that both 2. 3. 4. 5. 6. 7. RNA and antibodies may have catalytic activity, these are called ribozymes, and catalytic antibodies or abzymes, respectively) Biological catalysts, critical components of cell metabolism & biological processes. Very efficient catalysts Reduce G for reaction (by binding the transition state) Subject to regulatory control of various sorts Carry out catalysis in a special region of the molecule, the active-site Exhibit special kinetics Very efficient catalysts A very small quantity of an enzyme can catalyze the transformation of vastly lager quantity of the substrate Sucrase (invertase) can effect the hydrolysis of at least 1,000,000 times its own weight of sucrose without exhibiting any appreciable diminution in its activity Catalase is one of the more efficient enzymes, one molecule of this enzyme being able to catalyze the conversion 5,000,000 molecules of H202 per minute (the reduction of hydrogen peroxide to water and molecular oxygen) when conditions are favorable THE HYDROLISIS OF SUCROSE CATALYZED BY SUCRASE Kecepatan reaksi sederhana hidrasi karbon dioksida meningkat 107 kali lipat dengan enzim Carbonic anhydrase dibadingkan dengan tanpa enzim CO2 + H2O H2CO3 Setiap molekul Carbonic anhydrase dapat menghidrasi 105 molekul CO2 per detik Kecepatan reaksi dengan hexokinase 1010 phosphorylase 3.1011 alcohol dehydrogenase 2.108 creatine kinase 104. Enzymatic catalysts have much higher rates than non-enzymatic catalysts do, and even at relatively low temperatures (Table 1) UREA FERTILIZER The optimum conditions for enzyme catalysis are almost invariably moderate temperatures, and pHs which are not extreme The contrast between a reaction catalysed by an enzyme and by a nonenzymatic catalyst is well illustrated by the process of nitrogen fixation (i.e. reduction of N2 to ammonia). Nitrogenase catalyses this reaction at temperatures around 300 K and at neutral pH. The enzyme is a complex system comprising two dissociating protein components one of which contains iron and the other iron and molybdenum. Several molecules of ATP are hydrolyzed during the reduction By contrast, in the industrial synthesis of ammonia from nitrogen and hydrogen, the conditions used are as follows: temperature 700 - 900 K, pressure 100 - 900 atmospheres, and the presence of an iron catalyst, often promoted by traces of oxides of other metals 2. Enzyme Nomenclature Superfamilies: EC 1.1.3.4 Transferases Transfer functional groups between molecules Oxidoreductases Transfer electrons (RedOx reactions) Hydrolases Break bonds by adding H2O Lyases Elimination reactions to form double bonds Isomerases Intramolecular rearangements Ligases Join molecules with new bonds Enzyme Nomenclature 3. Enzyme Specificity The active site of an enzyme dictates specificity Tend to be specific for one type of chemical group Substrates have to interact in stereospecific manner (fit) Substrates have to bind relatively well (affinity) H-bonds, electrostatics, hydrophobicity Substrates have to react bonds to be broken or formed have to have proper reactivity Substances that fit and bind but don’t react are inhibitors Stereospecificity of Enzymes The specificity of enzymes for reactions is high different from inorganic catalysts. 1. Each enzyme catalyzes only one type of reaction, and in some cases will limit its activity to only one particular type of reactant molecule Incomparison, platinum catalyzes several different types of reactions. Enzim proteolitik, sebagai contoh, mengkatalisis hidrolisis ikatan peptida. Kebanyakan enzim proteolitik juga mengkatalisis reaksi yang berbeda tapi berhubungan yaitu hidrolisis ikatan ester Gambar 1. Hidrolisis ikatan peptida dan ester 2. Different end products are formed from the same substrate under the influence of different enzymes. The trisaccharide raffinose is hydrolized into melibiose and fructose in the presence of sucrase, while, the end products of the reaction are sucrose and galactose in the presence of emulsin Pyruvic acid is notable for the large number of compounds into which it may be converted by the action of different enzymes Sebagian enzim disintesis dalam bentuk yang tidak aktif, dan dikatifkan kemudian pada waktu dan tempat yang tepat secara fisiologi 3. This regulation is effected through changes in the catalytic activity of early enzymes in the pathway, carbamoyl-phosphate synthetase and aspartate carbamoyltransferase Enzim pencernaan seperti trypsinogen disintesis dalam pankreas dan diaktifkan oleh pemotongan ikatan peptida dalam usus (intestine) kecil untuk membentuk enzim aktif trypsin 4. The phenomenon of feedback inhibition is common in many biosynthetic pathways. In the biosynthetic pathway leading to the synthesis of pyrimidine nucleotidcs, the end products UTP and CTP (=Sitidintrifosfat) are able to inhibit the first enzyme in the pathway; thus they are able to limit the flow of metabolites into that pathway and so regulate their own biosynthesis Enzyme Active Sites The active site is the specific area of the enzyme to which the substrate attaches during the reaction The active site is part of the conformation of the enzyme molecule arranged to create a special pocket or cleft whose three-dimensional structure is complementary to the structure of the substrate The enzyme and the substrate molecules "recognize" each other through this structural complementarity The substrate binds to the enzyme through relatively weak forces -H bonds, ionic bonds (salt bridges), and van der Waals interactions between sterically complementary clusters of atoms. Lysozyme active site: Green shows substrate contacts and orange are catalytic residues Active site complements structure of substrate Contain amino acids that function in substrate binding, chemical catalysis, and product release Enzyme Three Dimensional Structure X-ray crystallography (also NMR); physical methods to solve structure of enzymes Conformation with or without substrate provides functional/ biological information Used to identify amino acids involved in catalysis • Example: Prostaglandin Synthase I with arachidonic acid • PGHS (COX) target of aspirin Enzyme Three Dimensional Structure 4. Enzyme Regulation Enzymes are tightly regulated light switches Unregulated enzymes become constitutively active or inactive (light is always on or off) Unregulated enzyme activity disrupts cell homeostasis and often lead to disease states. 5. Energi Aktivasi Kecepatan reaksi yang tinggi dengan keberadaan enzim berhubungan dengan energi aktivasi 1. Jika reaksi yang terjadi dalam sel berlangsung diluar sel, kecepatannya akan sangat lambat kecuali energi diberikan, misalnya dengan peningkatan suhu. Sementara reaksi dalam sel berlangsung pada suhu sekitar lingkungannya (mis. 5o - 40oC). ACTIVATION ENERGY 2. Kecepatan reaksi kimia yang tinggi pada suhu kamar (mis. laboratorium) tidak mungkin terjadi Karena kebanyakan reaksi kimia, sekalipun mengeluarkan energi, tidak terjadi secara spontan (berlangsung dengan sendirinya) tetapi membutuhkan tambahan energi yang disebut energi aktivasi (energy of activation). QUIZ 1. 2. 3. 4. 5. 6. 7. 8. 9. Define metabolism and anabolic and catabolic reactions Identify the parts of an enzymes catalyze reactions Describe method by which enzymes catalyze reactions Explain the lock-and-key and induced fit models of enzyme action Explain how changes in pH and temperature will affect enzyme activity Define vitamin, and explain why vitamins are essential for normal cellular function Describe the function of hormones in the living organism Define multienzyme system, and explain how such systems are regulated Describe the ways in which enzyme activity can be inhibited, and give examples of each type of inhibition