1 Biostatistics and Bioinformatics Biochemistry Modeling Biochemical Pathways Description of Module Subject Name Biochemistry Paper Name 13 Biostatistics and Bioinformatics Module Name/Title 16 Modeling Biochemical Pathways Dr. Vijaya Khader Dr. MC Varadaraj 2 Biostatistics and Bioinformatics Biochemistry Modeling Biochemical Pathways 1. Objectives: In this module, the students will understand 1. Biochemical Pathways and Pathways databases 2. Biochemical Pathway models and models databases 3. Simulating Biochemical Pathways 4. Modeling Biochemical Pathways using COPASI Brief Description Biochemical Pathways Biochemical Pathway Models Simulating Biochemical Pathway Modeling Biochemical Pathway Summary 2. Brief Description Dear students, Biochemistry is the study of enzyme catalysed reactions. The product of one enzyme catalyzed reaction may form the substrate of second enzyme catalysed reaction to yield a new product. In the second reaction, the product of first reaction acts as substrate to take the reaction forward to form another product. In addition, the product of the first reaction may bind to first enzyme to take the first reaction backward to form the first substrate. Further, the new product of the second reaction may form the substrate of third enzyme catalysed reaction and so on. Therefore, in this way, various substrates and products (collectively known as metabolites) are chained together in series, to yield, what we call the metabolic pathways. Consequently, metabolic pathway occurring within a cell is a series of enzyme catalysed chemical reactions starting with a substrate, chained together through intermediate metabolites to yield the final end product. The rate of turnover of these metabolites in a metabolic pathway is called Flux, or metabolic flux. 3 Biostatistics and Bioinformatics Biochemistry Modeling Biochemical Pathways In addition, the end product of one pathway may be used immediately to initiate another metabolic pathway. Therefore, all the metabolic pathways of a cell form an elaborate network of interconnected pathways, collectively known as Biochemical processes. The biochemical processes in the cell achieve biochemical phenotypes such as to meet energy requirements or to synthesize building blocks for growth or to undergo cell division or to become even a dormant cell such as spore. The metabolite flux of biochemical pathways is very vital for the survival of the cell under different conditions. The information about an enzyme catalysed reaction is collected using on-bench experiments and then it is used to develop a kinetic model of the enzyme, which is stored in reaction kinetics databases. The in vitro kinetic properties of the constituent enzymes of a biochemical pathway may be gathered from published literature and also from reaction kinetics databases. The in vitro kinetic properties of the constituent enzymes may then be used develop a model for a biochemical pathway. In case in vitro kinetic properties of some constituent enzymes is not available, then on-bench experiments may be conducted to collect in vitro kinetic properties of the remaining enzymes to develop a model. The in vivo behaviour of the pathway can then be understood in terms of the developed model. These models are deposited in databases and can be downloaded for use by other users to simulate pathways using simulation software. Consequently, once a model is available in the database, the same may be used to simulate metabolic pathway using simulation software to predict the behaviour of a biochemical process, without the need to conduct further on-bench experiments. Therefore, the objectives in this module are to learn Biochemical Pathways and databases storing information about Pathways. This will follow to learn Biochemical Pathway models and databases storing information about Pathway models. Simulation of Biochemical Pathways using COPASI will be undertaken for downloaded pathway model. Finally, we will learn using COPASI for modeling a biochemical pathways to gain insight into biochemical processes and phenotypes. Back to Concept Map 2.1. Metabolic Pathways Glycolysis was the first metabolic pathway discovered, which is used to meet the energy requirements of the cell. Metabolic pathways represent a series of metabolites transformed from the initial substrate or what we call the source metabolite, glucose-6-phosphate in glycolysis, to the end product of pathway, called the sink metabolite, the pyruvate. Technically all enzyme catalysed reactions are reversible. However, some reactions in a pathway may be highly exothermic resulting in irreversible reactions. Two reactions in glycolysis, the one catalysed by phosphofructokinase and the other catalysed by pyruvate 4 Biostatistics and Bioinformatics Biochemistry Modeling Biochemical Pathways kinase, are highly exothermic, and therefore irreversible. These two reactions make the glycolysis pathway as an irreversible pathway. In addition to metabolic pathways, there are pathways involving a group transfer reactions among several species, say several proteins. For example, a protein may receive a phosphoryl group, which is subsequently transferred to another protein. This phosphoryl group transfer may be repeated to transfer the phosphoryl group to some another protein. This chaining of phosphoryl transfers in series is called group transfer pathway. Taken together, metabolic pathways and group transfer pathways are collectively known as Biochemical pathways. The source metabolite i.e. first substrate and sink metabolite i.e. end product of the pathway are called boundary metabolites. The metabolites from substrate of second reaction to product of second last reaction are called intermediates. The source, the sink and the intermediate(s) are collectively known as metabolites. The first step to describe a biochemical pathway is to identify the start reaction and end reaction, which define the boundaries of the pathway. The glycolysis pathway shows that Glucosephosphate isomerase and Pyruvate kinase catalysed reactions, may be used to describe the boundaries of the glycolysis. 5 Biostatistics and Bioinformatics Biochemistry Modeling Biochemical Pathways KEGG database i.e. Kyoto Encyclopedia of Genes and Genomes, is a comprehensive resource of all known biochemical pathways. Visit KEGG at http://www.kegg.jp/kegg/pathway.html, and search for glycolysis by entering glycolysis followed by clicking Go button. However, the better way to search KEGG database is to search for a particular organism. Let us search the database for, say “Saccharomyces cerevisiae”. To do so, click on the Organism button. A new window will open, as shown next. Start typing the name of organism and select from the suggestions offered in the list. For the present example, select the third organism and click “Select” Button. Or in the Organism text box, simply enter the first alphabet of genus and two alphabets of species. For “Saccharomyces cerevisiae” enter ‘s’ for Saccharomyces and ‘ce’ for cerevisiae i.e. sce. After selecting Organism, enter the keyword “glycolysis” and click “Go” button. 6 Biostatistics and Bioinformatics Biochemistry Modeling Biochemical Pathways This will display search results in the browser window. Click on the first map i.e. thumbnail image This will open pathway map involving glycolysis in “Saccharomyces cerevisiae”. 7 Biostatistics and Bioinformatics Biochemistry Modeling Biochemical Pathways 8 Biostatistics and Bioinformatics Biochemistry Modeling Biochemical Pathways KEGG pathways connect metabolites in reactions using Enzyme Commission numbers. The ENZYME commission numbers on the pathway map are displayed in rectangles, such as 5.3.1.9 for Glucosephosphate isomerase. The metabolites are displayed as small circles labeled with names. Bringing the “mouse over” the metabolite circle, displays its structure, as shown for beta-D-Fructose 6-phosphate for the product of enzyme number 5.3.1.9 Different pathways are interconnected to form network of pathways. When The KEGG pathways database is searched for a particular organism, then the enzyme numbers are mapped on to the known genes in the genome of target organism. The mapping is displayed with ENZYME commission numbers highlighted with green background of the rectangle. This helps in understanding the biochemical processes in the target organism for the mapped genes of a pathway. To reach at the target gene entry, click on the highlighted rectangle, say 5.3.1.9, to reach the gene on the genome. 9 Biostatistics and Bioinformatics Biochemistry Modeling Biochemical Pathways This shows the mapping of glucose-6-phosphate isomerase enzyme to gene ‘YBR196C’ in the genome of “Saccharomyces cerevisiae” (i.e. budding yeast). In this way, one can reach to gene number in the genome and use the same for browsing gene in the genome browser as described in Module three “Molecular Sequence Databases”. Back to Concept Map 2.2. Biochemical Pathway Models Modeling of biochemical pathways, allows simulation of pathways for manipulation of various variables such as initial concentrations of metabolites. This manipulation results in prediction of the behavior of biochemical pathway, without actually working with the real cells. Therefore, the predictions from a model provide an easy way for testing hypotheses, before actually manipulating pathways within real cells. It allows the understanding and visualization of the biochemical pathway for manipulation in desired way. With a biochemical pathway model, one can actually visualize the metabolic