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Enzymology Dr. Nasir Jalal ASAB, National University of Sciences and Technology Course content Introduction and history of enzymes Historical aspects Discovery of enzymes Chemistry of enzymes Function and importance Enzymes in biotechnology Characteristics and properties Catalytic power and specificity Enzymes as catalysts ◦ ◦ Enzyme - substrate interactions Lock & key model ◦ Induced fit model ◦ ◦ Transition state model Quantum tunnelling model ◦ ◦ ◦ ◦ Hydrolase Lyase Isomerase ◦ Ligase Activity measurements Enzyme Purification and Assay Initial velocity measurements Assay types Enzyme units of activity Turnover number and properties Purification and assessment Methods for measurement Enzyme kinetics ◦ Michaelis-Menten Kinetics Enzymes as proteins Non-protein cofactors ◦ ◦ Introduction Assumptions Metal ions Organic cofactors ◦ ◦ ◦ Derivation Description of vo versus [S] Michaelis constant (KM) Nomenclature / Classification and Activity Measurements ◦ ◦ ◦ ◦ ◦ Oxidoreductase-dehydrogenase Transferase Course content ◦ Specificity/Substrate constant (SpC) ◦ Graphical Analysis of Kinetic Data, pH and Temp Dependence ◦ Graphical Analysis ◦ Substrate Binding Analysis ◦ Lineweaver-Burk Analysis ◦ Single Binding Site Model ◦ Hanes-Woolf Analysis ◦ Binding Data Plots ◦ Eadie-Hofstee Analysis ◦ Direct Plot ◦ Direct Linear Plot (Eisenthal/Cornish-Bowden Plot) ◦ Reciprocal Plot ◦ Nonlinear Curve Fitting ◦ Scatchard Plot ◦ pH-dependence of Michaelis-Menten Enzymes ◦ ◦ Temperature-Dependence of Enzyme Reactions Determination of Enzyme-Substrate Dissociation Constants Single Molecule Enzymology ◦ Kinetics ◦ ATP Synthase ◦ Equilibrium Dialysis ◦ ATP Synthase with Tethered Actin ◦ Equilibrium Gel Filtration ◦ Myosin-V ◦ Ultracentrifugation ◦ Kinesin motor attached to a fluorescent bead ◦ Spectroscopic Methods ◦ Single Molecule Studies of Cholesterol Oxidase Mechanism of enzyme catalysis ◦ β-galactosidase: a model Michaelis-Menten enzyme? Engineering More Stable Enzymes Incorporation of Non-natural Amino Acids into Enzymes Protein Engineering by Combinatorial Methods DNA Shuffling Enzyme inhibition and kinetics Classification of inhibitors ◦ Reversible, Irreversible, Iodoacetamide, DIFP Classification of Reversible Inhibitors Substrate Multi-substrate Reactions and Substrate Binding Analysis My office hours Thursdays Fridays [email protected] 9:00-11:00 2:00-4:00 Introduction to enzymology Enzymes are Biomolecules that catalyze, increase the rates of chemical reactions by 1015 to 1017 fold. Almost all enzymes are proteins. In enzymic reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts them into different molecules, the products. Living systems use enzymes to accelerate and control the rates of vitally important biochemical reactions. Brief history Earliest known use of enzymes comes from the Egyptian civilization which used yeast for fermentation and called the product Boza. HISTORY of Enzymes As early as the late 1700s and early 1800s, the digestion of meat by stomach secretions and the conversion of starch to sugars by plant extracts and saliva were known. However, the mechanism by which this occurred had not been identified. Fermentation In the 19th century, when studying the fermentation of sugar to alcohol by yeast, Louis Pasteur came to the conclusion that this fermentation was catalyzed by a vital force contained within the yeast cells called " ferments ", which were thought to function only within living organisms. He wrote that "alcoholic fermentation is an act correlated with the life and organization of the yeast cells, not with the death or putrefaction of the cells. Meaning of “enzyme” (Greek) In 1878 German physiologist Wilhelm Kühne (1837– 1900) first used the term enzyme , which comes from Greek ενζυμον (enzymon)"in leaven", to describe this process. The word enzyme was used later to refer to nonliving substances such as pepsin, and the word ferment used to refer to chemical activity produced by living organisms. In 1897 Eduard Buchner began to study the ability of yeast extracts that lacked any living yeast cells to ferment sugar. In a series of experiments at the University of Berlin, he found that the sugar was fermented even when there were no living yeast cells in the mixture. He named the enzyme that brought about the fermentation of sucrose "zymase". In 1907 he received the Nobel Prize in Chemistry“ for his biochemical research and his discovery of cell-free fermentation". History of Biotechnology Stages of Biotech evolution Ancient Classical Modern Ancient Biotechnology Ancient Biotech Begins with early civilization Developments in agriculture and food production Few records exist Ancient Biotech Archeologists research Ancient carvings and sketches sources of information Ancient Biotech Not known when biotech began exactly Focused on having food and other human needs Ancient Biotech Useful plants brought from the wild, planted near caves where people lived As food was available, ability to store and preserve emerged Ancient Food preservation most likely came from unplanned events such as a fire or freeze. Domestication 15,000 years ago, large animals were hard to capture People only had meat when they found a dead animal Came up with ways of capturing fish and small animals Domestication Food supplies often seasonal Winter food supplies may get quite low Domestication is seen by scientists as the beginning of biotech Domestication Adaptation of organisms so they can be cultured Most likely began 11,000 – 12,000 years ago in the middle east Domestication Involved the collecting of seed from useful plants and growing crude crops from that seed Involved the knowledge that the seed had to properly mature. A most recent find in Peru, documents the first civilization at 15000 years. Domestication Proper planting Need for water, light and other conditions for plant growth Earliest plants likely grains and other seeds used for food Domestication Raising animals in captivity began about the same time in history. Easier to have an animal close by than to hunt and capture a wild one. Domestication Learned that animals need food and water. Learned about simple breeding. How to raise young. Cattle, goats and sheep were the first domesticated food animals. Domestication About 10,000 years ago, people had learned enough about plants and animals to grow their own food The beginning of farming. Food Domestication resulted in food supplies being greater in certain times of the year. Products were gathered and stored. Food Some foods rotted Others changed form and continued to be good to eat Foods stored in a cool cave did not spoil as quickly Food Foods heated by fire also did not spoil as quickly Immersing in sour liquids prevented food decay Food preservation Using processes that prevent or slow spoilage Heating, cooling, keeps microorganisms (mo’s) from growing Food preservation Stored in bags of leather or jars of clay Fermentation occurs if certain mo’s are present Creates an acid condition that slows or prevents spoilage Classical Biotechnology Classical Biotech Follows ancient practices. Makes wide spread use of methods from ancient practices, especially fermentation. Methods adapted to industrial production e.g., salting, canning. Classical Biotech Produce large quantities of food products and other materials in short amount of time. Meet demands of increasing population. Many methods developed through classical biotech are widely used today. Cheese One of the first food products made through biotechnology Began some 4,000 years ago Nomadic tribes in Asia Cheese Strains of bacteria were added to milk Caused acid to form Resulting in sour milk Cheese Enzyme called “rennet” was added Rennet comes from the lining of the stomachs of calves Cheese Rennet is genetically engineered today. Not all cheese is made from produced rennet. Rennet is a complex of enzymes produced in mammalian stomach, and is used in the production of cheese. Rennet contains several enzymes, including the proteolytic enzyme protease that coagulates the milk, causing it to separate into solids (curds) and liquid (whey). They are also very important in the stomach of young mammals as they digest their mothers' milk. The active enzyme in rennet is called chymosin or rennin. Yeast Long used in food preparation and preservation Bread baking Yeast produces a gas in the dough causing the dough to rise Yeast Fermented products Vinegar Require the use of yeast stage of production. in at least one Sugar (glucose or fructose) → alcohol (ethanol) + carbon dioxide C6H12O6 → 2 CH3CH2OH + 2 CO2 Yeast Species of fungi Some are useful Some may cause diseases Vinegar Ancient product used to preserve food Juices and extracts from fruits and grains can be fermented Fermentation Process in which yeast enzymes chemically change compounds into alcohol In making vinegar the first product of fermentation is alcohol Fermentation Alcohol is converted to acetic acid by additional microbe activity Acid gives vinegar a sour taste Vinegar prevents growth of some bacteria Vinegar Keeps foods from spoiling Used in pickling Biblical references to wine indicate the use of fermentation some 3,000 years ago Fermentation control In ancient times, likely happened by accident Advancements occurred in the 1800’s and early 1900’s Fermenters Used to advance fermentation process Specially designed chamber that promotes fermentation Fermenters Allowed better control, especially with vinegar New products such as glycerol, acetone, and citric acid resulted Development Of yeasts that were predictable and readily available led to modern baking industry Modern Biotechnology Modern Biotech Manipulation of genetic material within organisms e.g., pruteen. Based on genetics and the use of microscopy, biochemical methods, related sciences and technologies. Modern Biotech Often known as genetic engineering Roots involved the investigation of genes Antibiotics Use of fermentation hastened the development of antibiotics A drug used to combat bacterial infections Antibiotics Penicillin Developed in the late1920’s Introduced in the 1940’s First drug produced by by microbes Antibiotics Many kinds available today Limitations in their use keep disease producing organisms from developing immunity to antibiotics Antibiotics Use antibiotics only when needed. Overuse may make the antibiotic ineffective when really needed later Antibiotics Some disease organisms are now resistant to certain antibiotics e.g., MDG Tuberculosis. Used in both human and veterinary medicine. Modern Biotech Deals with manipulating genetic info Microscopy and advanced computer technology are used In-depth knowledge of science Based on genetics research from the mid 1800’s Genetics Study of heredity Most work has focused on animal and plant genetics Genes – determiners of heredity Genes Carry the genetic code Understanding genetic structure essential for genetic engineering Heredity How traits are passed from parents to offspring Members of the same species pass the characteristics of that species Heredity Differences exist within each species. Differences are known as variability Heredity &variability Are used in modern biotechnology Sources of Variation include: 1. Independent assortment 2. Crossing over 3. Random fertilization Modern Biotech Use of biotech to produce new life forms Emerged in mid 1900’s Made possible by rDNA technology Research Use of systematic methods to answer questions. Problems may be basic or applied Basic Basic-- requires generating new info to gain understanding Applied – involve use of knowledge already acquired. Research Supplies facts that can be used to improve a process or product Settings range from elaborate labs to field plots Field Plot Small area of land that is used to test questions or hypothesis Belief is that same result would be obtained if carried out on larger scale. Often tested several times Known as replication Research Done by agencies, universities, private companies, individuals Biotech research in agriculture is carried out by experiment stations and large corporations. Development Creation of new products or methods based on findings of research Carefully studied before being put into full scale use Development New products tested before approval Government agencies such as the FDA are involved Prototype is developed – research model that is carefully tested Prototype Becomes a pattern for the production of similar products After being fully tested, full scale production begins. Specificity Almost all processes in a biological cell need enzymes in order to occur at significant rates. Enzymes are extremely specific for their substrates and as such speed up only specific reactions. There are no side or byproducts, which means that the products are also very specific. False discovery rate FDR Josh Elias from Stanford agrees. He says, “Partial tryptic search, or semi-tryptic searching, is ideal. And the reason for this is that it helps to distract away all the incorrect identifications”. Josh Elias from Stanford agrees. He says, “Partial tryptic search, or semi-tryptic searching, is ideal. And the reason for this is that it helps to distract away all the incorrect identifications”. this slide, which shows numbers of hits for a proteomics experiment against a target database and against a decoy database of assumed noise, using the target-decoy strategy presented in his talk and published in Elias & Gygi, “Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry”, Nature Methods – 4, 207 – 214 (2007). Catalytic power Most enzyme reaction rates are millions of times faster than those of comparable uncatalyzed reactions. As with all catalysts, enzymes are not consumed by the reactions they catalyze, nor do they alter the equilibrium of these reactions. However, enzymes do differ from most other catalysts by being much more specific. The ratio of uncatalyzed to catalyzed reaction rate is called the catalytic power. For uncatalyzed hydrolysis of urea the reaction rate is 3x104 and for catalyzed reaction it is 3X10-10 The Catalytic power is therefore 3x1014 . E-S Complex the concentration of substrate. Some enzymes are used commercially, for example, in the synthesis of antibiotics. In addition, some household products use enzymes to speed up biochemical reactions (e.g. , enzymes in biological washing powders break down protein or fat stains on clothes) Catalytic cycle of an enzyme
