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17.8: Enzymes - Biology

17.8: Enzymes - Biology


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Lab Objectives

At the conclusion of the lab, the student should be able to:

  • define the following terms: metabolism, reactant, product, substrate, enzyme, denature
  • describe what the active site of an enzyme is (be sure to include information regarding the relationship of the active site to the substrate)
  • describe the specific action of the enzyme catalase, include the substrate and products of the reaction
  • list what organelle catalase can be found in every plant or animal cell
  • list the factors that can affect the rate of a chemical reaction and enzyme activity
  • explain why enzymes have an optimal pH and temperature to ensure greatest activity (greatest functioning) of the enzyme (be sure to consider how virtually all enzymes are proteins and the impact that temperature and pH may have on protein function)
  • explain why the same type of chemical reaction performed at different temperatures revealed different results/enzyme activity
  • explain why warm temperatures (but not boiling) typically promote enzyme activity but cold temperature typically
  • decreases enzyme activity
  • explain why increasing enzyme concentration promotes enzyme activity
  • explain why the optimal pH of a particular enzyme promotes its activity
  • if given the optimal conditions for a particular enzyme, indicate which experimental conditions using that particular enzyme would show the greatest and least enzyme activity

Slideshow

A SlideShare element has been excluded from this version of the text. You can view it online here: pb.libretexts.org/bio1lm/?p=142

Introduction

Hydrogen peroxide is a toxic product of many chemical reactions that occur in living things. Although it is produced in small amounts, living things must detoxify this compound and break down hydrogen peroxide into water and oxygen, two non-harmful molecules. The organelle responsible for destroying hydrogen peroxide is the peroxisome using the enzyme catalase. Both plants and animals have peroxisomes with catalase. The catalase sample for today’s lab will be from a potato.

Enzymes

Enzymes speed the rate of chemical reactions. A catalyst is a chemical involved in, but not consumed in, a chemical reaction. Enzymes are proteins that catalyze biochemical reactions by lowering the activation energy necessary to break the chemical bonds in reactants and form new chemical bonds in the products. Catalysts bring reactants closer together in the appropriate orientation and weaken bonds, increasing the reaction rate. Without enzymes, chemical reactions would occur too slowly to sustain life.

The functionality of an enzyme is determined by the shape of the enzyme. The area in which bonds of the reactant(s) are broken is known as the active site. The reactants of enzyme catalyzed reactions are called substrates. The active site of an enzyme recognizes, confines, and orients the substrate in a particular direction.

Enzymes are substrate specific, meaning that they catalyze only specific reactions. For example, proteases (enzymes that break peptide bonds in proteins) will not work on starch (which is broken down by the enzyme amylase). Notice that both of these enzymes end in the suffix -ase. This suffix indicates that a molecule is an enzyme.

Environmental factors may affect the ability of enzymes to function. You will design a set of experiments to examine the effects of temperature, pH, and substrate concentration on the ability of enzymes to catalyze chemical reactions. In particular, you will be examining the effects of these environmental factors on the ability of catalase to convert H2O2 into H2O and O2.

The Scientific Method

As scientists, biologists apply the scientific method. Science is not simply a list of facts, but is an approach to understanding the world around us. It is use of the scientific method that differentiates science from other fields of study that attempt to improve our understanding of the world.

The scientific method is a systematic approach to problem solving. Although some argue that there is not one single scientific method, but a variety of methods; each of these approaches, whether explicit or not, tend to incorporate a few fundamental steps: observing, questioning, hypothesizing, predicting, testing, and interpreting results of the test. Sometimes the distinction between these steps is not always clear. This is particularly the case with hypotheses and predictions. But for our purposes, we will differentiate each of these steps in our applications of the scientific method.

You are already familiar with the steps of the scientific method from previous lab experiences. You will need to use your scientific method knowledge in today’s lab in creating hypotheses for each experiment, devising a protocol to test your hypothesis, and analyzing the results. Within the experimentation process it will be important to identify the independent variable, the dependent variable, and standardized variables for each experiment.

Part 1: Observe the Effects of Catalase

Procedure

  1. Obtain two test tubes and label one as A and one as B.
  2. Use your ruler to measure and mark on each test tube 1 cm from the bottom.
  3. Fill each of two test tubes with catalase (from the potato) to the 1 cm mark
  4. Add 10 drops of hydrogen peroxide to the tube marked A.
  5. Add 10 drops of distilled water to the tube marked B.
  6. Wait 60 seconds and measure the height of any bubbling you observe.
    1. Bubbling height tube A
    2. Bubbling height tube B
  7. What happened when H2O2 was added to the potato in test tube A?
  8. What caused this to happen?
  9. What happened in test tube B?
  10. What was the purpose of the water in tube B?

Part 2: Effects of pH, Temperature, and Substrate Concentration

Observations

From the introduction and your reading, you have some background knowledge on enzyme structure and function. You also just observed the effects of catalase on the reaction in which hydrogen peroxide breaks down into water and oxygen.

Questions

From the objectives of this lab, our questions are as follows:

  1. How does temperature affect the ability of enzymes to catalyze chemical reactions?
  2. How does pH affect the ability of enzymes to catalyze chemical reactions?
  3. What is the effect of substrate concentration on the rate of enzyme catalyzed reactions?

Hypotheses

Based on the questions above, come up with some possible hypotheses. These should be general, not specific, statements that are possible answers to your questions.

  • Temperature hypothesis
  • pH hypothesis
  • Substrate concentration hypothesis

Test Your Hypotheses

Based on your hypotheses, design a set of experiments to test your hypotheses. Use your original experiment to shape your ideas. You have the following materials available:

  • Test tubes
  • Catalase (from potato)
  • Hydrogen peroxide
  • Distilled water
  • Hot plate (for boiling water)
  • Ice
  • Acidic pH solution
  • Basic pH solution
  • Thermometer
  • Ruler and wax pencil

Write your procedure to test each hypothesis. You should have three procedures, one for each hypothesis. Make sure your instructor checks your procedures before you continue.

  • Procedure 1: Temperature
  • Procedure 2: pH
  • Procedure 3: Concentration

Results

Record your results—you may want to draw tables. Also record any observations you make. Interpret your results to draw conclusions.

  1. Do your results match your hypothesis for each experiment?
  2. Do the results reject or fail to reject your hypothesis and why?
  3. What might explain your results? If your results are different from your hypothesis, why might they differ? If the results matched your predictions, hypothesize some mechanisms behind what you have observed.

Communicating Your Findings

Scientists generally communicate their research findings in written reports. Save the things that you have done above. You will be use them to write a lab report a little later in the course.

Sections of a Lab Report

  • Title Page: The title describes the focus of the research. The title page should also include the student’s name, the lab instructor’s name, and the lab section.
  • Introduction: The introduction provides the reader with background information about the problem and provides the rationale for conducting the research. The introduction should incorporate and cite outside sources. You should avoid using websites and encyclopedias for this background information. The introduction should start with more broad and general statements that frame the research and become more specific, clearly stating your hypotheses near the end.
  • Methods: The methods section describes how the study was designed to test your hypotheses. This section should provide enough detail for someone to repeat your study. This section explains what you did. It should not be a bullet list of steps and materials used; nor should it read like a recipe that the reader is to follow. Typically this section is written in first person past tense in paragraph form since you conducted the experiment.
  • Results: This section provides a written description of the data in paragraph form. What was the most reaction? The least reaction? This section should also include numbered graphs or tables with descriptive titles. The objective is to present the data, not interpret the data. Do not discuss why something occurred, just state what occurred.
  • Discussion: In this section you interpret and critically evaluate your results. Generally, this section begins by reviewing your hypotheses and whether your data support your hypotheses. In describing conclusions that can be drawn from your research, it is important to include outside studies that help clarify your results. You should cite outside resources. What is most important about the research? What is the take-home message? The discussion section also includes ideas for further research and talks about potential sources of error. What could you improve if you conducted this experiment a second time?

Fumaric acid

Fumaric acid is an organic compound with the formula HO2CCH=CHCO2H. A white solid, fumaric acid occurs widely in nature. It has a fruit-like taste and has been used as a food additive. Its E number is E297. [3] The salts and esters are known as fumarates. Fumarate can also refer to the C
4 H
2 O 2−
4 ion (in solution). Fumaric acid is the trans isomer of butenedioic acid, while maleic acid is the cis isomer.

  • Fumaric acid
  • trans-1,2-Ethylenedicarboxylic acid
  • 2-Butenedioic acid
  • trans-Butenedioic acid
  • Allomaleic acid
  • Boletic acid
  • Donitic acid
  • Lichenic acid
  • 110-17-8 Y
  • CHEBI:18012 Y
  • ChEMBL503160 Y
  • 10197150 Y
  • DB04299 Y
  • C00122 Y
  • 88XHZ13131 Y
InChI=1S/C4H4O4/c5-3(6)1-2-4(7)8/h1-2H,(H,5,6)(H,7,8)/b2-1+ Y Key: VZCYOOQTPOCHFL-OWOJBTEDSA-N Y

Introduction

Synthetic biology is, according to a National Academy of Sciences (2013, 2) report of working parties from the US, the UK, and China, “an emerging discipline that combines both scientific and engineering approaches to the study and manipulation of biology.” Similar descriptions have been put forward by other commissions and studies. For example, a joint opinion by three scientific committees of the European Commission (Breitling et al. 2015) emphasizes the role of design and engineering approaches by stating that synthetic biology is “the application of science, technology and engineering to facilitate and accelerate the design, manufacture and/or modification of genetic materials in living organisms.” A report of the Secretariat of the Convention on Biological Diversity (2015) suggests that while there is no agreed international definition, the key features of synthetic biology include “the de novo synthesis of genetic material and an engineering-based approach to develop components, organisms and products.”

Proponents of synthetic biology suggest that its capabilities to design and redesign biological components and systems will address global food and energy challenges, propel industrial transformation as sustainable bio-engineered processes replace current petrochemical technologies, and offer new gene-based methods to target human medical conditions and insect-borne diseases (Church and Regis 2012 Weber and Fussenegger 2012 National Academies of Science 2013 Le Feuvre et al. 2016). The growth of synthetic biology has been boosted by a series of scientific and technological developments. These include improvements in DNA synthesis (longer fragments and higher accuracy), reduced DNA synthesis and sequencing costs, new capabilities not only to read but also to edit and rewrite the genes and cells of organisms, advances in bio-engineering design and modeling techniques, enhanced tools for biological assembly and engineering, the development of standardized biological parts, and the use of automated and data-intensive methods to speed up discovery and testing (Canton et al. 2008 Cheng and Lu 2012 Keasling 2012 Church et al. 2014 Lienert et al. 2014 Breitling and Takano 2015 Shih and Moraes 2016). The spread of synthetic biology has also been accelerated by targeted research programs and public policies, including funding by multiple federal agencies in the US (Wilson Center 2015 Si and Zhao 2016), by the UK’s network of synthetic biology research centers and its national synthetic biology roadmap (UK Synthetic Biology Roadmap Coordination Group 2012 Synthetic Biology Leadership Council 2016), by European Union projects (ERASynBio 2014), and by growing support in China (Synbiobeta 2016). Increased synthetic biology R&D investment and intellectual property acquisition by leading private sector companies in pharmaceutical, agricultural, chemical, and other sectors (OTI 2015 Carbonell et al. 2016), new business start-ups with ambitious goals such as cow-free milk or open-source insulin (Qiu 2014 Tucker 2015), community-based bio-hacking labs (Scudellari 2013), and the iGEM international synthetic biology competition (Kelwick et al. 2015) have also contributed to the emergence of the domain. At the same time, ethical, risk, equity, and other policy concerns and have been raised about the potential implications of applications of synthetic biology (Tucker and Zilinskas 2006 ETC Group 2010 OECD 2014 Engelhard 2016). These concerns have highlighted attention to the importance of responsible research and innovation in synthetic biology (Douglas and Stemerding 2013 Li et al. 2015 Shapira and Gök 2015).

In this context of rapid scientific advancement, increased public and private R&D, and stakeholder debate about the regulation and governance of synthetic biology, methods that can track the growth of research and innovation in synthetic biology are essential to inform engagement, policy deliberation, and management, and to provide evidence for decision-making. While there is a degree of high-level expert convergence on the conceptualization of synthetic biology, there are blurry boundaries between the technology in question, legacy technologies, and other new technologies that might be related to it (Nature Biotechnology 2009 Thomas 2014). There are epistemic debates about the distinctions between synthetic biology, systems biology, and genetic engineering (O’Malley et al. 2007 Calvert 2008). Synthetic biology has a legacy that extends back to the human genome project of the 1990s and early 2000s (Shapira et al. 2015) and earlier advances in understanding genes. At the same time, synthetic biology has relationships with advances in other disciplines, including engineering, biochemistry, agriculture, and informatics. Recent online discussions, hosted by the Biosafety Clearing House under the Convention on Biological Diversity (2015), demonstrate a range of perspectives from different countries and various stakeholders, about an operational definition of synthetic biology.

This paper puts forward a bibliometric approach to delineating synthetic biology. We recognize the broad notion that synthetic biology involves the design and engineering of biological components and systems at the genetic level. We also acknowledge that there is significant debate about details that affect the operationalization of a bibliometric definition of synthetic biology. We thus tread carefully through these debates, realizing that they are not yet resolved, to put forth a pragmatic strategy for creating a bibliometric definition of synthetic biology. There is relatively little work so far available on the bibliometric definition of synthetic biology, and our review of several of the definitions published to date finds them either too narrow or too expansive. We seek to contribute by refining an approach that better captures the complex scope of synthetic biology. We employ a multi-stage method, drawing from two publication indices (Web of Science and PubMed). The approach is used to identify scientific papers published in the synthetic biology domain and to trace patterns of emergence including international spread, funding, and disciplinary contributions.

The next section describes our search strategy and the steps and procedures involved. This is followed by a comparison of our results with those of other recent bibliometric definitions of synthetic biology and by an analysis of patterns of synthetic biology emergence indicated by the synthetic biology publications captured by our approach. The last part of the paper discusses the analysis and its limitations, draws conclusions and suggests lines for further work.


The sequencing data obtained in this study have been deposited to the NCBI Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE130399. Source data for Figs. 1e–g, 2b,e,f, 3a,b and 4b–d are available with the paper online. External data sets used in this study are available from GEO: ENCODE DNase-seq (GSE37074), PolyA-RNA-seq (GSE39524) of mouse NIH/3T3 cells, sci-CAR mixed cells datasets (GSE117089), SPLiT-seq (GSE110823), sci-RNA-seq (GSE98561), Drop-seq (GSE63269), sci-ATAC-seq (GSE67446), and dscATAC-seq (GSE123581) or from the 10X genomics website, 10X scRNA-seq (https://www.10xgenomics.com, 1k_hgmm_v3_nextgem dataset). All other data are available upon reasonable request.

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Author information

Ruiting Lin, Shannon Elf and Changliang Shan: These authors contributed equally to this work.

Affiliations

Department of Hematology and Medical Oncology, Winship Cancer Institute of Emory, Emory University School of Medicine, Atlanta, Georgia 30322, USA

Ruiting Lin, Shannon Elf, Changliang Shan, Hee-Bum Kang, Taro Hitosugi, Jae Ho Seo, Dongsheng Wang, Georgia Zhuo Chen, Sagar Lonial, Martha L. Arellano, Hanna J. Khoury, Fadlo R. Khuri, Sumin Kang, Jun Fan & Jing Chen

Department of Chemistry and Institute for Biophysical Dynamics, University of Chicago, Chicago, Illinois 60637, USA

Quanjiang Ji, Lu Zhou, Liang Zhang & Chuan He

Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA

Shuai Zhang, Daniel J. Brat & Keqiang Ye

Cell Signaling Technology, Inc. (CST), Danvers, Massachusetts 01923, USA

Jianxin Xie, Meghan Tucker & Ting-Lei Gu

Children’s Research Institute, UT Southwestern Medical Center, Dallas, Texas 75390, USA

Jessica Sudderth, Lei Jiang & Ralph J. DeBerardinis

Eugene McDermott Center for Human Growth and Development, UT Southwestern Medical Center, Dallas, Texas 75390, USA

Department of Chemistry, Emory University School of Medicine, Atlanta, Georgia 30322, USA

Department of Radiology, Emory University School of Medicine, Atlanta, Georgia 30322, USA

College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China

Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30322, USA

Novartis Institutes for BioMedical Research, Cambridge, Massachusetts 02139, USA

School of Basic Medical Sciences, Fudan University, Shanghai 200032, China

Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520, USA

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Contributions

R.L., S.E. and C.S. contributed equally to this work. J.X., T.-L.G., S.Z., K.Y., P.R.C., D.J.B., M.L.A., S.L., H.J.K., Q.L. and F.R.K. provided critical reagents. S.J.H. performed data analysis of pharmacokinetics studies. M.T. and T.-L.G. performed mass spectrometry-based assays. Q.J., L.Zhou, L.Zhang and C.H. performed biochemical analysis of lysine-acetylated 6PGD and molecular docking studies and analysed the data. J.S., L.J., M.M., R.J.D., S.W., Y.L. and H.M. performed quantitative mass spectrometry and NMR-based assays, and analysed data. B.H.L. performed the histopathological analyses. T.J.B. performed structural analyses. D.W. and G.Z.C. helped with xenograft experiments. C.S., S.E., H.-B.K., J.H.S., T.H. and J.F. performed all other experiments. R.L., S.E., C.S., S.K., J.F. and J.C. designed the study and wrote the paper. S.K., J.F. and J.C. are senior authors and jointly managed the project. All authors read and approved the final manuscript.

Corresponding authors


Abstract

Tomato (Lycopersicon esculentum) is one of the widely grown vegetables worldwide. Fusarium oxysporum f. sp. lycopersici (FOL) is the significant contributory pathogen of tomato vascular wilt. The initial symptoms of the disease appear in the lower leaves gradually, trail by wilting of the plants. It has been reported that FOL penetrates the tomato plant, colonizing and leaving the vascular tissue dark brown, and this discoloration extends to the apex, leading to the plants wilting, collapsing and dying. Therefore, it has been widely accepted that wilting caused by this fungus is the result of a combination of various physiological activities, including the accumulation of fungal mycelia in and around xylem, mycotoxin production, inactivation of host defense, and the production of tyloses however, wilting symptoms are variable. Therefore, the selection of molecular markers may be a more effective means of screening tomato races. Several studies on the detection of FOL have been carried out and have suggested the potency of the technique for diagnosing FOL. This review focuses on biology and variability of FOL, understanding and presenting a holistic picture of the vascular wilt disease of tomato in relation to disease model, biology, virulence. We conclude that genomic and proteomic approachesare greater tools for identification of informative candidates involved in pathogenicity, which can be considered as one of the approaches in managing the disease.


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Conclusions

We developed MESSA, a web server that integrates the results of a dozen state-of-the-art sequence analysis tools to provide predictions on local sequence properties, three-dimensional structure and function of a given protein. MESSA offers a user-friendly interface and display the results in a manner convenient for navigation. Our benchmark study showed that MESSA was able to offer extensive information for most of the proteins in a genome. We hope MESSA can help biologists to gain insights about proteins under study.


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