Laboratory Guide to Biochemistry, Enzymology, and Protein Physical Chemistry A Study of Aspartate Transcarbamylase
Laboratory Guide to Biochemistry, Enzymology, and Protein Physical Chemistry A Study of Aspartate Transcarbamylase Marc le Maire Centre National de la Recherche Scientifique Gif-sur-Yvette, France Raymond Chabaud Universite Pierre et Marie Curie Paris, France Guy Herve Centre National de la Recherche Scientifique Gif-sur-Yvette, France SPRINGER SCIENCE+BUSINESS MEDIA, LLC
This book is a translation, revised and enlarged by the authors, of Biochimie un modele d'etude: I'aspartate transcarbamylase theorie et guide d'experiences; published by Masson in Paris in 1990. ISBN 978-1-4613-6704-8 ISBN 978-1-4615-3820-2 (ebook) DOI 10.1007/978-1-4615-3820-2 1991 Springer Science+Business Media New York Originally published by Plenum Press New York in 1991 All rights reserved No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher
Foreword The study of a single well-chosen substance, here aspartate transcarbamylase, can provide an excellent basis for a laboratory course. The student is introduced to a variety of scientific ideas and to many experimental and interpretive techniques. This enzyme is readily available, is relatively stable, has an extensive literature, and its behavior has many facets: substrate inhibition, a large change in structure upon homotropic activation by substrates, allosteric stimulation by ATP, allosteric inhibition by CTP synergistic with VTP, positive cooperativity for substrates, negative cooperativity for CTP binding, and dissociation and reassembly of subunits C 3 and R2 from the holoenzyme C 6 I\5. In addition to the known biochemical aspects of these properties, the results obtained here can be interpreted in the light of the high-resolution X-ray diffraction structures of the T and R forms, the low-angle X-ray scattering results, and the large number of mutants now available by recombinant DNA methods. Future development of this course could also involve part of these methods, as well as the carefully chosen experiments described here. This approach resembles research more than the approaches one usually finds in biochemical laboratory courses. A consistent development of ideas about a single enzyme, which shows so many facets in its behavior, is sure to hold the interest of the student. Moreover, one explores a depth, and reasons to move forward, that are an essential part of research. Some years ago, I conceived and put into practice a somewhat similar plan for the laboratory part of a first semester of general chemistry: The student prepared a transition metal complex, made a chemical analysis, analyzed spectra (uv, visible, infrared, NMR), and then measv
vi Foreword ured kinetics of a subsequent transformation of the complex. This program was very successful in that it incorporated preparative, analytical, and physical-chemical methods as essential parts of a larger purpose. I expect similar success with the plan so well described in this guide. Harvard University William N. Lipscomb
Preface This book presents an integrated set of laboratory experiments devised for the teaching of biochemistry, enzymology, and protein physical chemistry. The program relies entirely on the use of a unique enzyme, aspartate transcarbamylase, which exhibits all of the catalytic and regulatory properties characteristic of allosteric enzymes. It can be easily and reversibly dissociated into subunits that have only the catalytic properties and thus behave as a simple Michaelian enzyme. A comprehensive study of this system leads to the use of numerous biochemical techniques. Biochemistry and molecular biology are young sciences, under rapid development. It is desirable that students of these subjects have the benefit of concrete training directly connected to today's practices as developed in research laboratories. Aspartate transcarbamylase is presently the object of high-level fundamental research, and the following teaching program benefits directly from the results obtained. An up-todate list of references guides the reader to more detailed information. The book begins with a general presentation of aspartate transcarbamylase and of the concepts of cooperativity and allostery. A study of the expression of the gene coding for the enzyme is then described, followed by a chapter dealing with the purification of the enzyme and its dissociation into subunits. The next chapter describes a wide experimental program concerning the structure and physical chemistry of the enzyme and its subunits. Finally, an extensive study of the catalytic and regulatory properties of these proteins is proposed. Each chapter begins with a presentation of the underlying theory. Appendixes provide useful information, including constants and units. Within each experimental section several questions are posed, and answers are given at the end of the book. vii
viii Preface Initially devised for the course Biochemistry and Cellular Biology and Physiology at the Pierre et Marie Curie University of Paris (Paris VI), this successful program is now used in other universities both in France and abroad. It is addressed primarily to undergraduate biochemistry students as well as to those following courses that involve biochemistry (medical schools; schools of pharmacy and agronomy). The book is sufficiently detailed to be of interest also to graduate students. It is a flexible program that can be adapted in terms of the number and nature of experiments performed in order to organize 50 to 200 hours of laboratory classes. Teachers will find at the end of the book a nonexhaustive list of complementary pedagogic possibilities that are offered by the system studied but are not presented in detail in this edition. The authors are at your disposal should you wish to examine closely any of these possibilities, as well as those emerging from current research. Criticisms and suggestions from our teaching colleagues or students are welcome. We wish to thank all of our friends who have already helped us with their advice and encouragement, in particular Dr. Anthony Else, who efficiently improved the English version of this book. We especially thank Ms. Jocelyne Mauger (Laboratoire d'enzymologie of CNRS) for her skillful collaboration in its framing. Gifsur-Yvette and Paris Marc Ie Maire Raymond Chabaud Guy Herve
Contents Chapter 1 ASPARTATE TRANSCARBAMYLASE 1. Structure of ATCase.................................... 2 2. Cooperativity between the Catalytic Sites.................. 8 3. Influence of the Effectors CTP, UTP, and ATP (PSE Mechanism)...................................... 12 Chapter 2 MOLECULAR GENETICS: REGULATION OF ASPARTATE TRANSCARBAMYLASE BIOSYNTHESIS 1. The ATCase Operon and Its Regulation... 17 2. Experimental Determinations: Measurement of the Lagtime for Derepression and Repression of ATCase Operon Expression... 19 2.1. Principle... 19 2.2. Bacterial Strain.................................... 19 2.3. Growth Medium... 19 2.4. Experimental Procedures... 20 Chapter 3 PURIFICATION OF ASPARTATE TRANSCARBAMYLASE AND ITS SUBUNITS 1. Purification of ATCase.................................. 25 1.1. Preparation of the Bacterial Extract................... 26 ix
x Contents 1.2. Heat Step... 26 1.3. Ammonium Sulfate Precipitation..................... 27 1.4. Ion Exchange Chromatography...................... 27 1.5. Precipitation at the Isoelectric Point.................. 28 2. Dissociation of ATCase into Catalytic and Regulatory Subunits... 28 2.1. Preparation of Catalytic Subunits by Heat Shock........ 28 2.2. Dissociation by p-hydroxymercuribenzoate............ 29 2.3. Dissociation by Neohydrin.......................... 31 3. Reconstitution of ATCase from Its Isolated Catalytic and Regulatory Subunits... 32 Chapter 4 STRUCTURAL AND PHYSICOCHEMICAL STUDY OF ASPARTATE TRANSCARBAMYLASE 1. Absorption Spectrophotometry and Determination of Protein Concentration.................................. 33 1.1. Theory... 33 1.2. Experimental Determinations: Assay of Protein Concentration... 37 2. Analysis by Electrophoresis... 39 2.1. Theoretical Aspects... 39 2.2. Experimental Determinations... 44 3. Analytical Ultracentrifugation............................ 57 3.1. Principle of Analytical Ultracentrifugation... 57 3.2. Sedimentation Velocity... 59 3.3. Sedimentation Equilibrium... 66 4. Size Exclusion Chromatography... 70 4.1. Principle... 70 4.2. The Stokes Radius................................ 71 4.3. Column Calibration................................ 74 Chapter 5 EN2YMATIC CATALYSIS AND REGULATION 1. Theoretical Aspects.................................... 91 1.1. Michaelian Enzymes... 91
Contents xi 1.2. Allosteric and Cooperative Enzymes... 105 2. Experimental Approach... 115 2.1. Measurement of the Enzymatic Activity by Incorporation of [I4C)-Aspartate... 116 2.2. Measurement of Enzymatic Activity by Using a Colorimetric Assay... 121 2.3. Enzymatic Properties of ATCase and Its Isolated Catalytic Subunits... 124 Chapter 6 COMPLEMENTARY EXPERIMENTS 1. Molecular Biology..................................... 141 2. Physical Chemistry... 141 3. Enzyme Kinetics... 142 APPENDIX 1. Equations and Units... 143 2. Molar Mass and Molecular Mass... 144 3. Units of Catalytic Activity... 145 4. Units of Radioactivity... 147 5. Units of Quantity... 147 6. k Factor of the Ultracentrifuge Rotor... 147 7. Calculation of Acceleration... 148 8. Bacterial Strains....................................... 148 9. Solutions and Reagents... 148 9.1. 0.8-M Tris-Acetate Buffer, ph 8... 148 9.2. 0.2-M Acetic Acid... 148 9.3. 2 x 10-3-M CTP... 149 9.4. 4 x 10-3-M CTP... 149 9.5. 2 x 10-2-M ATP... 149 9.6. Elution Buffer for Chromatography: lox Stock Solution... 149 9.7. 100-mM Aspartate, ph 8... 149 9.8. 10-mM Carbamyl aspartate, ph 8... 149 9.9. 1-M Phosphate Buffer, ph 7.2... 150 9.10. Buffer for Dilution of E... 150
xii Contents 9.11. Buffer for Dilution of C... 150 9.12. Buffer for Dilution of R and Recombination... 150 9.13. 200-mM Cacodylate Buffer, ph 6, 7, and 7.5... 150 9.14. 200-mM Tris-Acetate Buffer, ph 8 and 9... 150 9.15. 200-mM Glycine Buffer, ph 10... 151 9.16. 400-mM Succinate, ph 7... 151 9.17. Reaction Medium: 20-mM CAP-200-mM Tris- Acetate, ph 8... 151 10. Preparation of Standard Protein Mixtures for Column Calibration... 151 ANSWERS TO QUESTIONS... 153 REFERENCES... 159 INDEX... 167