Enzymology In Medical Biotechnology PDF Lecture Notes 2022/2023

ENZYMOLOGY IN MEDICAL BIOTECHNOLOGY Professor:Alessandra Astegno Academic year: 2022/2023 Università di Verona (Molecular and medical biotechnology) INDEX GENERAL INFORMATION ABOUT THE COURSE..................................................................................

ENZYMOLOGY IN MEDICAL BIOTECHNOLOGY Professor:Alessandra Astegno Academic year: 2022/2023 Università di Verona (Molecular and medical biotechnology) INDEX GENERAL INFORMATION ABOUT THE COURSE.................................................................................................. 1 INTRODUCTION: PROTEIN THERAPEUTICS........................................................................................................ 1 ENZYMATIC CATALYSIS #01.......................................................................................................................... 2 PRINCIPLES OF CATALYSIS #01.................................................................................................................. 4 ACTIVE SITE OF CATALYSTS........................................................................................................................ 4 ENZYMATIC CATALYSIS #02.......................................................................................................................... 5 PRINCIPLES OF CATALYSIS #02.................................................................................................................. 8 ENZYMATIC CATALYSIS #03........................................................................................................................ 10 PRINCIPLE OF CATALYSIS #03.................................................................................................................. 10 ENZYME KINETICS #01........................................................................................................................... 12 ENZYME CATALYSIS #04............................................................................................................................. 15 PRODUCTION OF RECOMBINANT PROTEINS (CLONING, EXPRESSION, AND PURIFICATION) #01.............................. 18 MOLECULAR CLONING #01..................................................................................................................... 19 PRODUCTION OF RECOMBINANT PROTEINS (CLONING, EXPRESSION, AND PURIFICATION) #02.............................. 21 MOLECULAR CLONING #02..................................................................................................................... 21 EXPRESSION HOST SYSTEM...................................................................................................................... 22 BACTERIAL CELL-BASED PROTEIN EXPRESSION......................................................................................... 22 YEAST CELL-BASED PROTEIN EXPRESSION................................................................................................ 24 INSECT CELL-BASED PROTEIN EXPRESSION............................................................................................... 24 PROTEIN PURIFICATION #01................................................................................................................... 25 LAB EXPERIENCES #01............................................................................................................................... 26 PRODUCTION OF RECOMBINANT PROTEINS (CLONING, EXPRESSION, AND PURIFICATION) #03.............................. 27 PROTEIN PURIFICATION #02................................................................................................................... 27 PROTEIN ENGINEERING #01........................................................................................................................ 30 RATIONAL DESIGN................................................................................................................................. 30 DIRECTED EVOLUTION #01..................................................................................................................... 33 PROTEIN ENGINEERING #02........................................................................................................................ 34 DIRECTED EVOLUTION #02..................................................................................................................... 34 CREATING LIBRARY DIVERSITY............................................................................................................... 35 ERROR PRONE PCR........................................................................................................................ 35 SITE SATURATION MUTAGENESIS (SSM)............................................................................................ 36 DNA SHUFFLING........................................................................................................................... 37 RANDOM-PRIMING RECOMBINATION (RPR)....................................................................................... 38 RACHITT (RANDOM CHIMERAGENESIS ON TRANSIENT TEMPLATES).......................................................... 38 ITCHY (ITERACTIVE TRUNCATION FOR THE CREATION OF HYBRID ENZYMES)............................................... 40 SCRATCHY..................................................................................................................................... 40 SCREENING PROCESS #01....................................................................................................................... 40  SELECTION #01................................................................................................................................. 41 PROTEIN ENGINEERING #03........................................................................................................................ 42 SELECTION PROCESS #02........................................................................................................................ 42 SELECTION #02................................................................................................................................. 42 SCREENING....................................................................................................................................... 44 ENZYME THERAPY IN METABOLIC DISORDERS #01.......................................................................................... 49 LYSOSOMAL STORAGE DISEASES (LSD)..................................................................................................... 50 SPHINGOLIPIDS.............................................................................................................................. 53 GAUCHER DISEASE #01...................................................................................................................... 54 ENZYME THERAPY IN METABOLIC DISORDERS #02.......................................................................................... 56 GAUCHER DISEASE #02...................................................................................................................... 56 POMPE DISEASE................................................................................................................................. 57 ENZYME THERAPY IN METABOLIC DISORDERS #03.......................................................................................... 61 ADENOSINE DEAMINASE (ADA) DEFICIENCY AND SEVERE COMBINED IMMUNODEFICIENCY DISEASE (SCID)..... 62 AMINO ACIDS................................................................................................................................ 67 PHENYLKETONURIA #01..................................................................................................................... 68 ENZYME THERAPY IN METABOLIC DISORDERS #04.......................................................................................... 69 PHENYLKETONURIA #02...................................................................................................................... 69 HOMOCYSTINURIA #01...................................................................................................................... 72 ENZYME THERAPY IN METABOLIC DISORDERS #05.......................................................................................... 74 HOMOCYSTINURIA #02...................................................................................................................... 74 ENZYME THERAPY IN METABOLIC DISORDERS #06.......................................................................................... 78 HOMOCYSTINURIA #03...................................................................................................................... 78 DRUG DEVELOPMENT AND CLINICAL TRIALS............................................................................................... 80 PRECLINICAL STUDIES......................................................................................................................... 80 CLINICAL STUDIES.............................................................................................................................. 80 CANCER TREATMENT #01........................................................................................................................... 82 AMINO ACID DEPLETION THERAPIES......................................................................................................... 83 ASPARAGINE DEPRIVATION #01........................................................................................................... 84 CANCER TREATMENT #02........................................................................................................................... 85 ASPARAGINE DEPRIVATION #02........................................................................................................... 86 ARGININE DEPRIVATION...................................................................................................................... 89 METHIONINE DEPRIVATION................................................................................................................. 93 FIBRINOLYTIC ENZYME FOR THROMBOLYTIC THERAPY..................................................................................... 94 PLASMINOGEN ACTIVATORS.................................................................................................................... 95 SECOND-GENERATION DRUG: TISSUE PLASMINOGEN ACTIVATOR................................................................ 97 FIRST-GENERATION DRUG: STREPTOKINASE............................................................................................ 97 THIRD-GENERATION DRUG: PROTEIN ENGINEERED OF TPA........................................................................ 99 ENZYMES IN DIAGNOSIS AND DIAGNOSTICS #01.......................................................................................... 101 THE CONTINUOUS ASSAYS................................................................................................................. 102 THE STOPPED ASSAYS....................................................................................................................... 103 DETERMINATION OF THE ENZYME VELOCITY......................................................................................... 104 ENZYME ACTIVITY............................................................................................................................ 105 ENZYMES IN DIAGNOSIS #01................................................................................................................. 105 LIVER ENZYMES............................................................................................................................... 106 ENZYMES IN DIAGNOSIS AND DIAGNOSTICS #02.......................................................................................... 109 ENZYMES IN DIAGNOSIS #02................................................................................................................. 109 MUSCLE ENZYMES........................................................................................................................... 109 ENZYMES IN DIAGNOSTICS #01............................................................................................................. 111 BLOOD OXIDASE (BLOOD GLUCOSE).................................................................................................... 111 UREASE (UREA MEASUREMENT)......................................................................................................... 112 LUCIFERASE #01.............................................................................................................................. 113 ENZYMES IN DIAGNOSIS AND DIAGNOSTICS #03.......................................................................................... 116 ENZYMES IN DIAGNOSTICS #02............................................................................................................. 116 LUCIFERASE #01.............................................................................................................................. 116 PEROXIDASES.................................................................................................................................. 120 APPLICATION OF PEROXIDASE IN ANALYSIS AND DIAGNOSTIC KITS......................................................... 120  07/03 GENERAL INFORMATION ABOUT THE COURSE The lab is mandatory, in which we will purify an enzyme, responsible for homocystinuria, WT and mutated one and it will be done some analysis of the difference between the WT and the mutated one. It is required to write a scientific report. The exam is a written exam with multiple choices and some open questions, that can include also some information about the lab experience. It can be asked also to draw some amino acid residues. INTRODUCTION: PROTEIN THERAPEUTICS Proteins can be developed and produced to serve specific therapeutic functions, such as treating metabolic disorders, resisting infections, arresting the spread of cancer, etc. These protein- based drugs can be grouped into the following based on their pharmacological activity: 1) Group 1: therapeutics with enzymatic or regulatory activity 2) Group 2: therapeutics with special targeting activity 3) Group 3: vaccines 4) Group 4: diagnostic agents Protein therapeutics have several advantages over small-molecule drugs: ◼ proteins often serve a highly specific and complex set of functions that cannot be mimicked by simple chemical compounds. ◼ because the action of proteins is highly specific, there is often less potential for protein therapeutics to interfere with normal biological processes and cause adverse effects. ◼ because the body naturally produces many of the proteins that are used as therapeutics, these agents are often well tolerated and are less likely to elicit immune responses (they are less immunogenic). ◼ for diseases in which a gene is mutated or deleted, protein therapeutics can provide effective replacement treatment without the need for gene therapy, which is not currently available for most genetic disorders. ◼ the clinical development and FDA approval time of protein therapeutics may be faster than that of small-molecule drugs. A study published in 2003 showed that the average clinical development and approval time was more than 1 year faster for 33 protein therapeutics approved between 1980 and 2002 than for 294 small-molecule drugs approved during the same time period. ◼ because proteins are unique in form and function, companies are able to obtain far-reaching patent protection for protein therapeutics. The first recombinant protein used in therapy was insulin, that is a hormone with protein nature that in its absence there is the development of diabetes, that if it is not treated, it can cause severe consequences. In 1922 insulin was purified for the first time from a bovine pancreas, because it could save the life of diabetic patients. Nowadays, it is known that the purification of proteins from the natural source has many problems, as it has an impact in the number of the source, the cost of 1 the purification, as well as developing immunological issues. In 1978 it was produced the first engineered insulin from E. coli bacteria, which overcomes some of these problems encountered. However, the recombinant protein is powerful, but it presents many challenges: a) once produced the protein, it should be stored, distributed, understood the properties of the proteins in terms of solubility, ability to enter the body, but also stability. One major protein problem is the degradation of the proteins, through lysosome or proteosome, but also by the action of the proteases. b) Immune response, as the immune response can neutralise the effect of the introduced protein but also can have severe effect upon introduction of the protein. c) It is required the administration of an active protein/enzyme, in the active conformation as there are many denaturing factors that can disturb the active form. But it should be taken in consideration also the post-translational modification (bacteria don’t have post-translational mechanisms, but mammals and baculovirus yes), as they are able to regulate the activity of the protein. It should also take in consideration also the number of proteins while taking in consideration the post-translational modifications (it should be taken an equilibrium, a compromise between the number of active proteins on the total of proteins extracted). d) Producing a recombinant protein not only takes a long time, passing through the different phases of drug production, but it is also an expensive procedure. e) Ethical problems: the government has an amount of money that can decide where to invest it for the research for the treatment of rare diseases or the treatment of diseases that are widely distribute in the population like cancer, neurodegenerative diseases (Alzheimer disease, Parkinson disease, etc.), etc. This can cause some ethical issues as the small population has the same possibility to have a treatment and it is up to the government to decide where to invest the money. ENZYMATIC CATALYSIS #01 It is hard to overstate the huge importance of enzyme to the biological organisms. Not only because they are diffused in the organisms, but also because they are responsible for metabolic chemical. The metabolic reactions require catalysis. The metabolic reaction must occur very fast to sustain life (10-5-102 s). Many chemical reactions are slow (many reactions can occur spontaneously and in a fast way, but there are also some reactions, like the glycine decarboxylation, can take a very long time to occur in isolation). Catalysts can accelerate reactions without heating. The catalysts can be ions, but also biological material. Enzymes are biological catalysts, made of proteins. Why are enzymes used as catalysts? 1) The enzymes are efficient, with acceleration rates of up to 17 orders of magnitudes. 2) The enzymes are substrate-specific, as the active site complements the substrate geometrically and electrostatically. They are specific because they are proteins that have the 3D structure. Some enzymes are specifics, as they can distinguish an enantiomer from others (they are enantiospecific, which is very useful industrial production, like in drug production). 2 3) The enzymes are reaction-specific, catalysing a single reaction. There are 6 classes of enzymes: a. Oxidation-reduction class, that catalyses the oxidoreduction reaction. b. Group transfer class, that catalyses the transfer a chemical group from one molecule to the other. c. Hydrolysis class, that catalyses the reaction in which the molecule is broke down using a water molecule. d. Water-independent lysis class, that catalyses the reaction hydrolysis of a molecule without a water molecule. e. Isomerisation class, that catalyse the isomerisation of a chemical group or a double bond. f. Ligation class, which catalyses the bound of 2 molecules, the ligation. These reactions are a complex reaction, which is unfavourable. Usually, the ligation is coupled by the consumption of energy, by hydrolysing ATP) Scientists found a way to classify enzymes, using the enzyme classification (E.C). The E.C includes the assignment of 4-digit numbers, where the first indicated the class of enzyme and the other numbers indicates specific information on the reaction. For example, the reaction of transfer of amidino group from arginine to glycine is catalysed by the enzyme encoded by E.C=2.1.4.2, where ➔ 2 indicated the class of transferases. ➔ 1 indicates that it is transferred a group containing 1 C atom. ➔ 4 indicates that it is transferred an amidino group. ➔ 2 indicated that the acceptor molecule is glycine. With the E.C number it is possible to identify the enzyme and its specific reaction. 4) Enzymes can couple energy-demanding reactions to ATP hydrolysis, which metal catalysts cannot do. The coupling is done by phosphate transfer from ATP to one of the substrates and its destabilisation. 5) Enzymes can be regulated, as they are proteins. The regulation can be done by the following means: o Small molecules (competition, allostery) o Phosphorylation, as example of posttranslational modifications (that include acetylation, glycosylation, SUMOylation, ubiquitination, etc.) o Degradation o Synthesis 6) Certain enzymes can create large molecular machines for carrying out complex tasks, that cannot be done by the single components of the supramolecular complexes. Examples: a. DNA polymerase (genome replication) b. RNA polymerase (gene expression) c. Spliceosome (gene expression) d. Ribosome (protein translation) 3 PRINCIPLES OF CATALYSIS #01 The chemical reactions covert reactants to products through a high-energy transition state. During the reaction, there is a drop in the free energy, which means that the reaction is favourable (ΔG