Introduction to

Subtopics

 Science,  What is mathematics?  Demarcation problem  Classification of Science  Definition and scope of biochemistry  Historical perspective  Applications and careers in biochemistry  What is life?  Chemical Vs. biochemical reactions  Hierarchical organization

Science and Scientific method

What is Science?

 Science is an intellectual and practical activity encompassing the systematic study of the structure and behavior of the physical and the natural world through observation and experiment.  Science is the pursuit and application of knowledge and understanding of the natural and social world following a systematic methodology based on evidence.  Science is a body of knowledge attained through the scientific method.

The scientific method

 The scientific method is a set of principles and procedures for the systematic pursuit of knowledge involving the recognition and formulation of a problem, the collection of data through observation and experiment, and the formulation and testing of hypotheses.  Scientific method is an empirical and systematic method of acquiring knowledge based on evidence.  The scientific method is a continuous process that has five basic elements. These elements can be illustrated by the discovery of the structure of DNA. These are 1. Problem identification:

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The scientific method begins with defining a question or a particular problem.

Examples:

 Why is the sky blue?  How can we design a drug to cure Corona Virus?  How can we stop expansion of the Desert Locust?  Previous investigation of DNA had determined its chemical composition (the four nucleotides), the structure of each individual nucleotide, and other properties. In 1950 Gregor Mendel showed that genetic inheritance had a mathematical description. DNA had been identified as the carrier of genetic information by the Avery–MacLeod–McCarty experiment in 1944 ('s transforming principle) but the mechanism of how genetic information was stored in DNA was unclear. 2. Formulating a hypothesis

A hypothesis is a proposed explanation suggesting a possible correlation between or among a set of phenomena made on the basis of limited evidence as a starting point for further investigation. A hypothesis is a suggested explanation of a phenomenon, or alternately a reasoned proposal. Formulating a hypothesis involves induction based on previous observations. The hypothesis to be tested is called statistical or alternative hypothesis. The null hypothesis states the alternative hypothesis is false. Scientists want to show that the null hypothesis is false. A scientific hypothesis must be falsifiable, implying that it is possible to identify a possible outcome of an experiment or observation that conflicts with predictions deduced from the hypothesis.

Examples:

 The scattering of blue light by air molecules make the sky blue.  Poultry drugs can be modified for human use to fight corona virus.  Toxic plants can be used to poison the Desert Locust.  Equivalence principle formulated by Albert Einstein  Linus Pauling proposed that DNA might be a triple helix. This hypothesis was also considered by and James D. Watson but discarded. Crick and Watson hypothesized that DNA had a double helical structure.  DNA makes RNA makes protein formulated by Francis Crick

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3. Derive predictions Predictions are specific but logical consequences of the formulated hypothesis. Predictions are deductions drawn from the hypotheses. Predictions must be able to distinguish the hypothesis from other likely hypotheses.

 Prediction: If DNA had a helical structure, its X-ray diffraction pattern would be X-shaped. This prediction was determined using the mathematics of the helix transform, which had been derived by Cochran, Crick and Vand (and independently by Stokes). This prediction was a mathematical construct, completely independent from the biological problem at hand. 4. Test predictions: Hypothesis testing involves conducting experimental and measurement-based testing to check the validity of the predictions. Evidences are generated by empirical experiments. The purpose of an experiment is to determine whether observations agree with or conflict with the predictions derived from a hypothesis. Experiments should be designed to minimize possible errors, especially through the use of appropriate scientific controls. For example, tests of medical treatments are commonly run as double-blind tests. Personnel are unaware of the recipients of the desired test drugs and placebos. Hypothesis testing involves careful observation, applying rigorous skepticism about what is observed, given that cognitive assumptions can distort how one interprets the observation.

 Experiment: Rosalind Franklin crystallized pure DNA and performed X-ray diffraction to produce photo. The results showed an X-shape. Watson and Crick showed an initial (and incorrect) proposal for the structure of DNA to a team from Kings College – Rosalind Franklin, Maurice Wilkins, and Raymond Gosling. Franklin immediately spotted the flaws which concerned the water content. Later Watson saw Franklin's detailed X-ray diffraction images which showed an X-shape and was able to confirm the structure was helical. This rekindled Watson and Crick's model building and led to the correct structure. 5. Analyze the findings to draw conclusions

Analysis is done to compare the predictions of the alternative hypothesis to those of the null hypotheses. Various statistical tests are employed to draw conclusions leading to refinement (or elimination) of the hypotheses based on the experimental findings.

 Analysis: When Watson saw the detailed diffraction pattern, he immediately recognized it as a helix. He and Crick then produced their model, using this information along with the previously

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known information about DNA's composition and about molecular interactions such as hydrogen bonds.  Watson and Crick were able to infer the essential structure of DNA by concrete modeling of the physical shapes of the nucleotides which comprise it. They were guided by the bond lengths which had been deduced by Linus Pauling and by Rosalind Franklin's X-ray diffraction images. 6. Report the results

The findings are published and communicated to other scientists through a process of peer review for cross-checking and reproducibility. The paper describing the structure of DNA was published in 1953. The discovery became the starting point for many further studies involving the genetic material, such as the field of molecular genetics, and it was awarded the Nobel Prize in 1962.

Properties of scientific method

 Objective observation: Measurement and data (possibly although not necessarily using mathematics as a tool)  Reproducible evidences: repetition of experiment and/or observation as benchmarks for testing hypotheses  Induction: reasoning to establish general rules or conclusions drawn from facts or examples  Verification and testing: critical analysis, critical exposure to scrutiny, peer review and assessment

Mathematics

 According to Galileo Galilei, the father of modern science, the laws of nature are written in the language of mathematics… measure what is measurable and make measurable what is not so.  Mathematics is a language of nature and a language of science but it is not science.  Mathematics is a conceptual framework for logical comprehension of the natural world  Mathematics use patterns to formulate other patterns through its own proof.

The demarcation problem

Demarcation problem refers to properties to distinguish scientific beliefs from non-scientific beliefs (religious beliefs, wishful-thinking and artistic imagination). There three basic properties to distinguish scientific knowledge from non-scientific beliefs.

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1. Logical positivism: scientific beliefs are based on empirically verifiable statements. 2. Falsification: scientific beliefs are base on statements that can be proved false and refuted by invalidating observations or arguments. Science is open to falsification. 3. Uncertainty: scientific measurements are usually accompanied by estimates of their uncertainty. The scientific method is iterative. At any stage it is possible to refine its accuracy and precision.

Classification of science

Scientific fields are broadly divided into natural sciences (the study of natural phenomena) and social sciences (the study of human behavior and society). Natural sciences are further divided into biology, chemistry, physics and earth sciences.

Biochemistry

As the name indicates, biochemistry is an interdisciplinary science. Biology is the science of living organisms and it so far has no first principle. The unifying principle in biology is the theory evolution by natural selection. Chemistry is the science of atoms and molecules and the first principle in chemistry is the quantum theory of matter.

Biochemistry is the science of the atoms and molecules as governed by the laws of quantum chemistry in living organisms which are unified by the theory of evolution. Biochemistry is the chemistry of life processes; it deals with chemical processes within and related to living organisms. It studies the structure, composition, and chemical reactions of substances in living systems.

Scope of biochemistry

Historically, classical biochemists were a succession of hunters; microbiologists were microbe hunters, where as biochemists were vitamin hunters followed by enzyme hunters and and crystal hunters. Contemporary biochemistry has four main fields of specialization. These are: enzymolgy, structural biology, metabolism and molecular genetics.

Enzymology is the study of the properties, reaction kinetics, and catalytic mechanisms of enzymes. Enzymes, the reaction catalysts of biological systems, are central to every biochemical process. Enzymes have extraordinary catalytic power, high degree of specificity for their substrates, they accelerate

5 | P a g e chemical reactions tremendously, and they function in aqueous solutions under very mild conditions of temperature and pH.

Metabolism: is the study of the set of life-sustaining enzyme-catalyzed chemical reactions taking place within living organisms. It is concerned with how living things synthesize and degrade carbohydrates, lipids, amino acids, and nucleotides accompanied with generation and consumption of ATP (Energy). Metabolism is required for the conversion of food into energy, the conversion of small molecules from one to another and to bigger molecules and elimination of wastes. For example, the conversion of glucose into pyruvate through glycolysis is metabolism. Metabolic biochemists aim to ellucidate in the individual enzyme-catalyzed steps for the conversion of one substance to another.

Molecular genetics also known as molecular biology deals with the expression of genetic information and mechanism of regulation of cellular functions through genetic regulatory networks. It studies about the structure and function of nucleic acids. Molecular biologists are gene hunters; they aim to isolate a gene and characterize its physiological function.

Structural biology is the study of the molecular structure of biological macromolecules. It is concerned with the relationship between molecular structure and biochemical function. Structural biologists solve the three-dimensional structure enzymes, transport proteins, receptors and antibodies to understand the molecular mechanism of their actions. The major classes of biological molecules are proteins, nucleic acids, carbohydrates, and lipids. Structural biologists are crystallographers; they aim to isolate a protein and solve its structure to characterize its biochemical functions.

Historical perspectives

Year People Nations Discoveries 2012 Jenifer Doudna American CRISPR/CAS9 based genome editing is based on a French simplified version of the bacterial CRISPR-Cas9 antiviral Feng Zhang Chinese defense system. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing to be removed and/or new ones added in vivo. 1985 Kary Mullis American He invented the process known as polymerase chain reaction (PCR), in which a small amount of DNA can be

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copied in large quantities over a short period of time. 1972 Paul Berg American He succeeded in inserting DNA from a bacterium into the virus' DNA. He thereby created the first DNA molecule made of parts from different organisms. This type of molecule became known as "hybrid DNA" or "recombinant DNA". The birth of recombinant DNA technology or biotechnology. 1966 Marshall Nirenberg American Genetic codes unveiled: They showed the order Heinrich Matthaei German of nucleotides in nucleic acids, which carry the genetic Gobind Khorana Indian code of the cell and control the cell's synthesis of proteins. They determine the amino acid sequences of the proteins from the sequence of nucleic acids. 1959 John Kendrew English The determination of the first 3-D structure of protein Max Perutz Austrian those of sperm whale myoglobin by John Kendrew in 1959 and of human deoxyhemoglobin and horse methemoglobin by Max Perutz shortly thereafter, ushered in a revolution structural biochemistry. 1953 James D. Watson American They determined the structure of the DNA molecule. This Francis Crick Briton structure - a long double helix - contains a long row of pairs of four different nitrogen bases, which allow the molecule to function like a code. The molecule's structure also explains how it is able to copy itself. The nitrogen bases always pair in the same constellations, so that if a molecule is split, its halves can be supplemented so that they form copies of the original molecule. 1944 Oswald Avery Canadian Avery, MacLeod, and McCarty showed DNA to The Avery– Colin MacLeod Canadian MacLeod–McCarty experiment demonstrated that DNA is Maclyn McCarty American the agent of genetic transformation. It is the substance that causes bacterial transformations. It been widely believed that the genetic information was carried by proteins (with the very word protein itself coined to

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indicate a belief that its function was primary). They purify and characterize the "transforming principle" responsible for the transformation phenomenon first described in Griffith's experiment. 1937 Hans Krebs German He presented a complete picture of an important part of metabolism - the citric acid cycle. In this process, which has several steps, nutrients are converted to other molecules with a large amount of chemical energy. This is one of the most important achievements of metabolic biochemistry. 1926 James Sumner American He crystallized the first enzyme, jack bean urease, which catalyzes the hydrolysis of urea to NH3 and CO2 and demonstrated that these crystals consist of protein. 1918 Gustav Embden German They discovered most frequent type of glycolysis found in Otto Meyerhof, German the body that follows the Embden-Meyerhof-Parnas (EMP) Jakob Karol Parnas Polish Pathway. Mapped of the conversion of sugar to lactic acid in frog muscles. 1897 Eduard Buchner Swiss He obtained a cell-free yeast extract that could carry out the synthesis of ethanol from glucose (alcoholic fermentation). Regarded as the birth of modern biochemistry. 1894 Emil Fischer German He mapped the structure of glucose and discovered that glycolytic enzymes can distinguish between stereoisomeric sugars. He formulated the lock-and-key hypothesis: The specificity of an enzyme (the lock) for its substrate (the key) arises from their geometrically complementary shapes. 1869 Friedrich Miescher German He isolated phosphate rich chemical (nucleic acids) from the nuclei of the pus (white-blood cells) from infections. 1842 Justus Von Liebig Discovered the chemical theory of metabolism. Wrote on animal chemistry or organic chemistry and its application to physiology and medicine. 1828 Friedrich Wöhler German Wöhler synthesis of urea (organic molecule waste product

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of animal metabolism) from ammonium cyanate (inorganic mineral). He disproved the belief of vitalism. The belief assumed organic molecules (molecules found in living organisms) can only be produced by living organisms and could not be produced in the laboratory. The presence of a vital force distinguishes the living organic world from the non-living inorganic world. Vitalism is the belief that living organisms are fundamentally different from non-living entities because they contain some non-physical element or are governed by different principles than are inanimate things 1772 Antoine Lavoisier French Oxygen theory of combustion, combustion of a candle is similar to animal respiration as both need oxygen. Physiological process with reference to nonliving mechanism. Applications of Biochemistry

Medical sciences

AIDS is caused by HIV. HIV follows a series of steps to multiply inside human body. These are binding, fusion, reverse transcription, integration, replication, assembly and budding. AZT (Azidothymidine) also known as Zidovudine (ZDV) is a thymidine structural analogue. The 3’ hydroxyl group of thymidine is replaced with azide anion group. AZT is the first drug to gain approval for AIDS treatment from FDA. It selectively inhibits HIV’s reverse transriptase, the enzyme responsible for making a DNA copy from its RNA. HAART is the use of multiple drugs that act at different stages of the HIV life-cycle. These are entry inhibitors, reverse transcriptase inhibitors, integrase inhibitors and protease inhibitors. Similarly, scientists are working to develop drug against the nCOV.

Agricultural sciences

The insecticidal property of a soil-bacterium was discovered by its devastating effect on large populations of silkworms. Bacillus thuringensis naturally produces a toxin that kills many herbivorous insects. Researchers discovered that the main insecticidal activity was due to crystalline protein called delta endotoxin. The crystal structure, the biochemistry and mode of action of Bt toxin was thoroughly

9 | P a g e studied. Soon, Bt products were used efficiently for killing insects in organic farming. This was followed by introduction of the gene that encodes the toxic protein into plants. The first genetically engineered plant was Bt-corn. It is insect-resistant corn.

Food sciences

Purines are group of is nitrogenous compounds with important biological functions. Caffeine is a purine naturally found in coffee (Coffea arabicaa). Theophylline is a purine naturally found in tea (Camellia sinensis). Theobromine is a purine naturally found in cacao plant (Theobroma cacao). Caffeine acts both as antagonist of adenosine receptors in the brain and non-selective phosphodiesterase inhibitor. Binding of adenosine to adenosine receptors increase drowsiness and coffee antagonizes adenosine to maintain alertness. PDE enzymes degrade and regulate secondary messenger cAMP which in turn regulates the activity of protein kinase A (PKA). PKA initiates a cascade of phosphorylation reactions that shuts down glycogen synthesis and activates glycogen breakdown. Human brain is very energy demanding organ and glucose is the principal energy substrate of the brain.

Cathine is a phenetylamine type substance naturally found Khat (Catha edulis) which stimulates the CNS by functioning as full agonists of GPCR and serotonin the chemical messenger and neurotransmitter for happiness. Khat is consumed to achieve a state of euphoria. Drink warm milk before bed to get better sleep. Milk is very rich in tryptophan which is a precursor for synthesis of serotonin and melatonin. Melatonin is a hormone that regulates sleep-wake cycle. Tryptophan deficiency can result in lower serotonin level and mood disorder. Chocolate is rich in phenylalanine which is a precursor for dopamine. Dopamine is a neurotransmitter that can regulate mood. Some foods are fortified with vitamins and minerals to increase their quality.

Careers in biochemistry

 Ethiopian standard agency  Ethiopian petroleum supply enterprise  Ethiopian pharmaceutical supply agency  Ethiopian public health research institute  Armour Hannsen Research Institute  Ethiopian food and drug administration  Ethiopian environmental protection authority

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 Ethiopian institute of biotechnology  Biochemistry teaching in more than 40 Ethiopian universities  Forensic medicine  Toxicology

What is life?

Biology is the study of life and biochemistry is the study of the chemistry of life but what is life?

Aristotle was the first to attempt to define life. According to Aristotle 347 BC

 Life is anything that grows and maintains itself (through nutrition), and reproduces.

According to cell theory developed by Matthias Schleiden, Theodor Schwann and Rudolph Virchow in 1837- 1838

 All living organisms are composed of cells, and the cell is the basic structural and functional unit of life.

According Erwin Schrödinger in 1944

 Life is maintaining a stream of orderly events which escapes the decay into atomic chaos (disorder) and equilibrium.

According to information theory proposed by Claude E. Shannon in 1948

 Life is a naturally emergent property of a system of intimately interdependent parts communicating with each other using a molecular code for the purpose of self replication.

According to NASA’s astrobiology program in 1990, Adaptability is shared by all life on earth.

 Life is a self-sustaining chemical system capable of Darwinian evolution.

According to Norman Horowitz

 Life possesses the properties of replication, catalysis and mutability.

According to Edward Trifonov in 2011

 Life is self-reproduction with variations.

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According to popular definition

 Organisms are open systems that maintain homeostasis, are composed of cells, have a life cycle, undergo metabolism, can grow, adapt to their environment, respond to stimuli, reproduce and evolve.

Living things are extra ordinarily diverse. This diversity is the source of the problem with most proposed definitions of life: definitions tend of have loopholes and it is easy to find exception to rule. Hence, there is no unequivocal definition for all life forms.

Synthia is a partially synthetic bacterium in which synthetic bacterial genome was transplanted into DNA free cells.

The seven wonders of biology

1. Theory of evolution by natural selection is formulated by Charles Darwin in 1859 as an explanation for adaptation and speciation. Natural selection is a principle by which each slight variation of a trait is preserved if useful. The basic tenets of the theory are: i. More individuals are produced at each generation than the environment support and that can survive ii. Phenotypic variations exist among individuals and variation is heritable iii. Individuals with heritable traits better suited to the environment will survive (survival of the fittest iv. New species will form through reproductive isolation. 2. The cell theory was developed by Matthias Schleiden, Theodor Schwann and Rudolph Virchow in 1837- 1838. The three tenets of the cell theory are i. All living organisms are made up of cells ii. Cells are the basic structural and functional units of life iii. Cells arise from pre-existing cells.

Modern version of cell theory includes

iv. Energy flow (metabolism and biochemistry) occurs within cells governed by the laws of thermodynamics v. Heredity information (DNA) is passed from cell to cell vi. All cells have the same basic chemical composition

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3. The gene theory of heredity: The principles that govern heredity were discovered by Gregor Mendel in 1860’s. The main concept of the gene theory is that traits are passed from parent to offspring through gene transmission. Mendel discovered heritable factors or heritable units as the basic particles of heredity. These principles are called Mendel’s law of segregation and law of independent assortment. i. The law of segregation states that heritable factors are segregated during gamete formation ii. The law of independent assortment states that segregated heritable factors are randomly united at fertilization. Modern version of gene theory includes iii. Genes are segments of DNA located on chromosomes. 4. The discovery of chromosomes: Theodor Boveri, Thomas Hunt Morgan and others definitively demonstrated that chromosomes are the vectors of heredity. Genes are stored in chromosomes inside cell nuclei. 5. The discovery of DNA as genetic material: The Avery–MacLeod–McCarty experiment was an experimental demonstration that DNA is the substance that causes bacterial transformation in Griffith’s experiment. DNA serves the function of carrying genetic information and it is the chemical basis of heredity. 6. The double helical structure of DNA: The three-dimensional double helical model for the structure of DNA was proposed by James D Watson and Francis C. Crick in 1953. The structure elucidated the mechanism of base pairing by which genetic information is stored and copied. 7. The discovery of the genetic code and the central dogma of molecular biology: In 1961 Francis Crick introduced the central dogma of molecular biology in which DNA makes RNA makes protein. Marshall Nirenberg and Heinrich Matthaei produced a long RNA chain consisting of a single nucleotide. When this resulted in a long chain of a single amino acid, the first part of the genetic code "puzzle" fell into place.

Chemical and biochemical reactions

Organic reactions often require elevated temperatures and pressures, extremes of pH, high concentrations of reactants as well as organic solvents. Organic reactions cannot be easily stopped once started. In contrast, biochemical reactions require relatively mild conditions, nearly neutral pH and dilute aqueous solutions. Biochemical reactions are mediated by remarkable biological catalysts known

13 | P a g e as enzymes. The rates of biochemical reactions are so tightly regulated. Most biological reactions function as one step of a sequence of reactions that are organized into metabolic pathways coupled together by a universal biological energy “currency,” called ATP. ATP is produced from adenosine diphosphate (ADP) and a phosphate ion by energy generating processes. Although living organisms are enormously diverse in their macroscopic properties, there is a remarkable similarity in their biochemistry. Moreover, the series of metabolic pathways, as well as the structures of the enzymes that catalyze them are, for many basic processes, nearly identical from organism to organism. This strongly suggests that all known life-forms are descended from a single primordial common ancestor in which these biochemical features first developed.

Example: The combustion of glucose into flames producing carbon dioxide, water and energy is a chemical reaction. Once glucose starts burning, it cannot be stopped easily and all its energy is released at once. On the contrary, the combustion of glucose by living organisms is highly regulated. Energy is released step by step and the released energy is captured and stored in the form of ATP. Metabolic processes occur in temperatures below 100°C and atmospheric pressure. The concentration of both reactants and products is very low. Water is the solvent of most biochemical reactions.

Hierarchical organization

Biochemical unity and structural regularity underlies biological diversity. Structural regularity is derived from structural hierarchy. The smallest constituent of life is the atom. Atoms are combined to form a molecule. Many small molecules combine together to make up a macromolecule. Organized clusters of macromolecules make up supramolecular assemblies which form organelles. Subcellular organelles form a cell. A group of cells function together as a tissue and different tissues make up an organ. Organs work together to form an organ system of a multicellular organism. A group of individuals of the same species living together in an area is a population. Two or more populations interacting with each other form a community. Communities interacting with each other and with the physical environment encompass an ecosystem. The biosphere is the area of life on earth comprising all of the ecosystems.

Living organisms contain only a few types of biomolecules: water, proteins (Greek: proteios, of first importance; a term coined in 1838 by Jacob Berzelius), nucleic acids, polysaccharides (Greek: sakcharon, sugar) and lipids (Greek: lipos fat). Biological macromolecules are polymeric molecules exhibiting modular construction; they consist of linked monomeric units that occupy the lowest level of their structural hierarchy. Macromolecules include proteins (polymers of amino acids), nucleic acids

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(polymers of nucleotides), and polysaccharides (polymers of sugars). Proteins are synthesized from the same 20 species of amino acids, nucleic acids are made from 8 types of nucleotides (4 each in DNA and RNA), and there are 8 commonly occurring types of sugars in polysaccharides. The fourth major class of biomolecules is that of lipids. Lipids are too small to be classified as macromolecules but also have a modular construction.

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