Understanding enzymes: functionPDF电子书下载

PART Ⅰ ENZYME FUNCTION 3

1 A Short Practical Guide to the Quantitative Analysis of Engineered Enzymes&Christopher D.Bayer and Florian Hollfelder 3

1.1 Introduction 3

1.2 Quantifying Reaction Progress 4

1.3 Typical Saturation Plots Give Michaelis-Menten Parameters 5

1.4 What Can Go Wrong？ 8

1.5 Dealing with Multiphasic and Pre-Steady-State Kinetics 12

1.6 Evaluating Enzymes 16

2 Protein Conformational Motions：Enzyme Catalysis&Xinyi Huang，C.Tony Liu，and Stephen J.Benkovic 21

2.1 Introduction 21

2.2 Multidimensional Protein Landscape and the Timescales of Motions 22

2.3 Conformational Changes in Enzyme-Substrate Interactions 26

2.4 Conformational Changes in Catalysis 28

2.4.1 Protein Dynamics of DHFR in the Catalytic Cycle 30

2.4.2 Temporally Overlap：Correlation Does Not Mean Causation 32

2.4.3 Fast Timescale Conformational Fluctuations 34

2.4.4 Effect of Conformational Changes on the Electrostatic Environment 36

2.5 Conservation of Protein Motions in Evolution 38

2.6 Designing Protein Dynamics 39

2.7 Concluding Remarks 40

3 Enzymology Meets Nanotechnology：Single-Molecule Methods for Observing Enzyme Kinetics in Real Time&Kerstin G.Blank，Anna A.Wasiel，and Alan E.Rowan 47

3.1 Introduction 48

3.2 Single-Turnover Detection 53

3.2.1 Fluorescent Reporter Systems 53

3.2.2 Measurement Setup 56

3.2.3 Data Analysis 57

3.3 Single-Enzyme Kinetics 60

3.3.1 Candida antarctica Lipase B 63

3.3.2 Thermomyces lanuginosus Lipase 67

3.3.3 α-Chymotrypsin 73

3.3.4 Nitrite Reductase 78

3.3.5 Summary 84

3.4 New Developments Facilitated by Nanotechnology 88

3.4.1 Nano-optical Approaches 89

3.4.2 Nano-electronic Approaches 96

3.4.3 Nanomechanical Approaches 103

3.4.4 Summary 108

3.5 Conclusion 110

4 Interfacial Enzyme Function Visualized Using Neutron，X-Ray，and Light-Scattering Methods&Hanna Wacklin and Tommy Nylander 125

4.1 Phospholipase A2：An Interfacially Activated Enzyme 126

4.1.1 Neutron Reflection 129

4.1.2 Ellipsometry 130

4.1.3 Activity of Naja mossambica mossambica PLA2 130

4.1.4 Fate of the Reaction Products 133

4.1.5 The Lag Phase and Activation of Pancreatic PLA2 135

4.1.6 Distribution of Products during the Lag Phase 138

4.1.7 Hydrolysis of DPPC by Pancreatic PLA2 139

4.1.8 Role of the Reaction Products in PLA2 Activation 141

4.1.9 Effect of pH and Activation by Me-β-cyclodextrin 144

4.2 Other Lipolytic Enzyme Reactions on Surfaces 150

4.2.1 Triacylglycerol Lipases and the Role of Lipid Liquid Crystalline Nanostructures 150

4.3 Cellulase Enzymes 154

4.4 Conclusion 158

5 Folding Dynamics and Structural Basis of the Enzyme Mechanism of Ubiquitin C-Terminal Hydroylases&Shang-Te Danny Hsu 167

5.1 Introduction 169

5.1.1 UCH-L1 171

5.1.1.1 Genetic association between UCH-L1 and neurodegenerative diseases 171

5.1.1.2 UCH-L1 in oncogenesis 175

5.1.2 Molecular Insights into the Pathogenesis Associated with UCH-L1 175

5.1.3 UCHL3 177

5.1.4 UCHL5 178

5.1.5 BAP1 179

5.2 UCH Structures 180

5.3 Folding Dynamics and Kinetics 183

5.4 Substrate Recognition 184

5.5 Enzyme Mechanism 186

5.6 Conclusion 189

6 Stabilization of Enzymes by Metal Binding：Structures of Two Alkalophilic Bacillus Subtilases and Analysis of the Second Metal-Binding Site of the Subtilase Family&Jan Dohnalek，Katherine E.McAuley，Andrzej M.Brzozowski，Peter R.φstergaard，Allan Svendsen，and Keith S.Wilson 203

6.1 Introduction：Subtilases and Metal Binding 203

6.1.1 Calcium-Binding Sites in Bacillus：Proposal for a Standard Nomenclature 209

6.1.2 The Weak Metal-Binding Site 214

6.2 Two New Structures of Subtilases with Altered Calcium Sites 216

6.2.1 Proteinase SubTY 216

6.2.1.1 The overall fold 216

6.2.1.2 The active site 216

6.2.1.3 SubTY calcium and sodium sites 218

6.2.1.4 SubTY disulfide bridge 219

6.2.2 SubHal 220

6.2.2.1 The unliganded form of SubHal 220

6.2.2.2 The SubHal：CI2A complex 221

6.2.2.3 Termini，surface，and pH stability of SubHal 221

6.2.2.4 The two crystallographically independent SubHal：CI2A complexes 223

6.2.2.5 The calcium sites in SubHal 224

6.2.2.6 The active site of SubHal 226

6.2.3 Enzymatic Activity of SubTY and SubHal 228

6.2.4 Comparison of SubTY and SubHal with Other Subtilases 228

6.2.5 The SubHal C-domain Compared to the Eukaryotic PCs，Furin and Kexin 232

6.2.5.1 Active site comparison 233

6.2.5.2 The specificity pockets 234

6.2.5.3 Inhibitor CI2A binding 234

6.2.6 Activity Profiles 236

6.2.7 Comparison of Metal Binding at the Strong and Weak Sites in the S8 Family 236

6.2.8 The Ca-Ⅱ and Na-Ⅱ Metal-Binding Sites 237

6.3 Conclusion：Implications for Structural Studies of Enzymes 248

6.4 Materials and Methods 249

6.4.1 SubTY 249

6.4.1.1 Protein production and purification 249

6.4.1.2 Purification of the SubTY：CI2A （1：1）complex 250

6.4.1.3 Crystallization 250

6.4.1.4 Structure determination 2516.4.2 SubHal 251

6.4.2.1 Protein production and purification 251

6.4.2.2 Purification of the SubHal：CI2A （1：1）complex 252

6.4.2.3 Crystallization 252

6.4.2.4 Structure determination 253

6.4.3 Protease Assays 256

6.4.4 pH Stability 257

6.4.5 Data Deposition 257

7 Structure and Functional Roles of Surface Binding Sites in Amylolytic Enzymes&Darrell Cockburn and Birte Svensson 267

7.1 Introduction 267

7.2 Identifiication of SBSs：X-Ray Crystallography 271

7.3 Bioinformatics of SBS Enzymes 273

7.4 Binding Site Isolation 275

7.5 Protection of Binding Sites from Chemical Labeling 277

7.6 Nuclear Magnetic Resonance 277

7.7 Binding Assays 278

7.8 Activity Assays 282

7.9 Future Prospects 283

7.10 Conclusion 286

8 Interfacial Enzymes and Their Interactions with Surfaces：Molecular Simulation Studies&Nathalie Willems，Mickael Lelimousin，Heidi Koldsφ，and Mark S.P.Sansom 297

8.1 Introduction 297

8.2 Enzyme Interactions at Interfaces 299

8.3 Molecular Dynamic Simulations of Biomolecular Systems 301

8.4 Lipases 303

8.4.1 Atomistic MD Studies of Lipase Interactions with Interfaces 304

8.4.2 The Role of Water in Lipase Catalysis at Interfaces 307

8.5 Coarse-Grained MD Studies of Interfacial Enzymes：Orientation and Interactions 309

8.5.1 Phospholipase A2 309

8.5.2 PTEN 310

8.6 Conclusions 311

PART Ⅱ ENZYME DESIGN 321

9 Sequence，Structure，Function：What We Learn from Analyzing Protein Families&Michael Widmann and Jurgen Pleiss 321

9.1 Introduction 321

9.2 Detection of Inconsistencies Utilizing a Standard Numbering Scheme 323

9.3 Identification of Functionally Relevant Positions 327

9.4 The Modular Structure of Thiamine Diphosphate-Dependent Decarboxylases 330

9.5 Stereoselectivity-Determining Positions：The S-Pocket Concept in Thiamine Diphosphate-Dependent Decarboxylases 333

9.6 Regioselectivity-Determining Positions：Design of Smart Cytochrome P450 Monooxygenase Libraries 336

9.7 Substrate Specificity-Determining Positions：The GX/GGGX Motif in Lipases 340

9.8 Conclusion 341

10 Bioinformatic Analysis of Protein Families to Select Function-Related Variable Positions&Dmitry Suplatov，Evgeny Kirilin，and Vytas Svedas 351

10.1 Introduction 352

10.2 Bioinformatic Analysis of Evolutionary Information to Identify Function-Related Variable Positions 359

10.2.1 Problem Definition 359

10.2.2 Scoring Schemes in the Variable Position Selection：High-Entropy，Subfamily-Specific，and Co-Evolving Positions 361

10.2.3 Association of the Variable Positions with Functional Subfamilies 366

10.2.4 How to Select Functionally Important Positions as Hotspots for Further Evaluation：Implementation of Statistical Analysis 366

10.3 The Bioinformatic Analysis of Diverse Protein Superfamilies 369

10.3.1 Bioinformatic Challenges at Studying Enzymes 369

10.3.2 Zebra：A New Algorithm to Select Functionally Important Subfamily-Specific Positions from Sequence and Structural Data 370

10.4 Subfamily-Specific Positions as a Tool for Enzyme Engineering 375

10.5 Conclusion 377

11 Decoding Life Secrets in Sequences by Chemicals&Zizhang Zhang 387

11.1 Introduction 388

11.2 Linking an Enzyme’s Activity to Its Sequence 389

11.3 Refiining the Sequence Space to a Specifiic Function by Directed Evolution 395

11.4 Linking Chemistry to -Omics with High-Throughput Screening Methods 398

11.5 Finding Large Sequence Space of a Specific Function from Microbial Diversity 400

11.6 Linking Sequences to Substromes at the Molecular Level 404

11.6.1 Biocatalytic Study of EHs 405

11.6.2 Pharmacological Study of EHs 407

11.6.3 Mechanistic Study of EHs 407

11.6.4 What We Have Learned from the Studies of EH 410

11.6.5 Technologies with Potentials in Genochemistry Approach 410

11.7 Correlating with Computational Methods 410

11.8 Problems That Genochemistry Can Potentially Tackle 413

11.9 Conclusion 41412 Role of Tunnels and Gates in Enzymatic Catalysis&Sergio M.Marques，Jan Brezovsky，and Jiri Damborsky 421

12.1 Introduction 421

12.2 Protein Tunnels 423

12.2.1 Structural Basis and Function 423

12.2.2 Identification Methods 427

12.2.3 Molecular Engineering 429

12.3 Protein Gates 431

12.3.1 Structural Basis and Function 431

12.3.2 Identification Methods 437

12.3.3 Molecular Engineering 440

12.4 Conclusions 442

13 Molecular Descriptors for the Structural Analysis of Enzyme Active Sites&Valerio Ferrario，Lydia Siragusa，Cynthia Ebert，Gabriele Cruciani，and Lucia Gardossia 465

13.1 Introduction：Molecular Descriptors for Investigation of Enzyme Catalysis 465

13.2 Molecular Descriptors Based on Molecular Interaction Fields 467

13.3 Multivariate Statistical Analysis for Processing and Interpretation of Molecular Descriptors 472

13.4 Grind Descriptors for the Study of Substrate Specificity 475

13.5 VolSurf Descriptors for the Modeling of Substrate Specifiicity 477

13.6 Differential MIFs Descriptors for the Study of Enantioselectivity 479

13.7 Hybrid MIFs Descriptors for the Computation of Entropic Contribution to Enantiodiscrimination 481

13.8 Analysis of Enzyme Active Sites for Rational Enzyme Engineering 484

13.9 BioGPS Descriptors for in silico Rational Design and Screening of Enzymes 489

13.10 Conclusions 495

14 Hydration Effects on Enzyme Properties in Nonaqueous Media Analyzed by MD Simulations&Diana Lousa，Antonio M.Baptista，and Claudio M.Soares 501

14.1 Enzyme Reactions in Nonaqueous Solvents 502

14.2 Classes of Nonaqueous Solvents 503

14.3 The Role of Water in Nonaqueous Biocatalysis 504

14.4 Effect of Water Content on Enzyme Structure and Dynamics 504

14.5 Effect of Water Content on Enzyme Selectivity 507

14.6 Hydration Mechanisms of Enzymes in Polar and Nonpolar Solvents 508

14.7 Enzyme Behavior as a Function of Water Activity 510

14.8 Hydration Effects on Enzyme Reactions in Ionic Liquids 512

14.9 Hydration Effects on Enzyme Reactions in Supercritical Fluids 514

14.10 Conclusions 516

15 Understanding Esterase and Amidase Reaction Specificities by Molecular Modeling&Per-Olof Syren 523

15.1 Introduction 523

15.2 Fundamental Catalytic Concepts 525

15.2.1 Fundamental Chemistry of Amides and Esters 525

15.2.2 Esterases and Amidases and Their Metabolic Significance 525

15.2.3 Fundamental Chemical Aspects of Amidase and Esterase Catalysis 526

15.2.4 Impact of Stereoelectronic Effects on the Enzymatic Reaction Mechanism 529

15.3 Molecular Modeling of Fundamental Catalytic Concepts 529

15.3.1 QM Calculations on Amidases and Esterases 529

15.3.2 MD Simulations on Amidases and Esterases 535

15.3.3 QM/MM Simulations on Amidases and Esterases 539

15.4 Outlook and Implications for Enzyme Design 544

15.5 Additional Comments 546

PART Ⅲ ENZYME DIVERSITY 561

16 Toward New Nonnatural TIM-Barrel Enzymes Using Computational Design and Directed Evolution Approaches&Mirja Krause and Rik K.Wierenga 561

16.1 Introduction 562

16.2 General Aspects of Protein Engineering 566

16.2.1 Library Creation Methods 569

16.2.2 Structure-Based Library Design 572

16.2.3 Optimal Libraries for Directed Evolution Methods 574

16.2.4 Data-Driven Design （Semirational Design） 578

16.2.5 Protein Engineering by Selection and Screening Methods 579

16.3 Directed Evolution Studies with TIM-Barrel Enzymes 584

16.3.1 Protein Engineering Studies of TIM-Barrel Proteins 586

16.3.2 The Kemp Eliminases 590

16.4 Concluding Remarks 596

17 Handling the Numbers Problem in Directed Evolution&Carlos G.Acevedo-Rocha and Manfred T.Reetz 613

17.1 Introduction 614

17.2 Saturation Mutagenesis in Directed Evolution 617

17.3 Statistical Analyses 620

17.3.1 Conventional Statistics Based on the Patrick and Firth Algorithm 620

17.3.2 Statistics Based on the Nov Algorithm 624

17.4 How to Group and Randomize Amino Acid Positions 626

17.5 Fitness Landscapes 628

17.5.1 Fujiyama vs.Badlands Fitness Landscapes 628

17.5.2 Fitness-Pathway Landscapes and How to Escape from Local Minima 630

17.6 Conclusions and Perspectives 636

18 Hints from Nature：Metagenomics in Enzyme Engineering&Esther Gabor，Birgit Heinze，and Jurgen Eck 643

18.1 Metagenomics and the Ideal Enzyme 644

18.2 Molecular Microdiversity 647

18.3 Metagenomic Enzyme Chimera 650

18.4 Outlook 653

19 A Functional and Structural Assessment of Circularly Permuted Bacillus circulans Xylanase and Candida antarctica Lipase B&Stephan Reitinger and Ying Yu 657

19.1 Introduction 657

19.2 Naturally Occurring Circular Permutations：Selected Examples 658

19.3 Circular Permutation of Bacillus circulans Xylanase 661

19.4 Circular Permutation on Candida antarctica Lipase B 669

19.5 Conclusion 674

20 Ancestral Reconstruction of Enzymes&Satoshi Akanuma and Akihiko Yamagishi 683

20.1 Introduction 683

20.2 Reconstruction of an Ancestral Protein Sequence 684

20.2.1 Overview 684

20.2.2 Methods for Ancestral Sequence Reconstruction 684

20.2.3 Early Works 686

20.3 The Commonote 687

20.3.1 The Last Universal Common Ancestor，the Commonote 687

20.3.2 Theoretical Studies on the Environmental Temperature of the Commonote 688

20.3.3 Reconstruction of an Ancestral Nucleoside Diphosphate Kinase 689

20.3.4 Estimation of the Environmental Temperature of the Commonote 692

20.4 Application to Designing Thermally Stable Proteins 693

20.4.1 Design of Thermally Stable Proteins 693

20.4.2 Case Studies to Create Thermally Stable Enzymes by Introducing Ancestral Residues as Amino Acid Substitutions 694

20.4.3 Reconstruction of Thermally Stable，Ancestral DNA Gyrase Using a Small Set of Homologous Amino Acid Sequences 696

20.5 Conclusion 697

PART Ⅳ ENZYME SCREENING AND ANALYSIS 707

21 High-Throughput Screening or Selection Methods for Evolutionary Enzyme Engineering&Shuobo Shi，Hongfang Zhang，Ee Lui Ang，and Huimin Zhao 707

21.1 Introduction 708

21.2 Selection 710

21.2.1 Solid-Medium-Based Selection 717

21.2.2 Liquid-Medium-Based Selection 719

21.2.3 Display-Based Selection 722

21.3 Screening 724

21.3.1 Chromatography- and Mass-Spectrometry-Based Screening 725

21.3.2 Solid-Medium-Based Screening 726

21.3.3 Microtiter-Plate-Based Screening 727

21.3.4 Yeast Two-/Three-Hybrid System 729

21.3.5 FACS-Based Screening 729

21.3.6 Microfluidics-Based Screening 732

21.4 Conclusions and Prospects 734

22 Nanoscale Enzyme Screening Technologies&Helen Webb-Thomasen and Andreas H.Kunding 745

22.1 Introduction 745

22.2 Approaches to Nanocompartmentalization of Enzymes 746

22.2.1 Liposomes 747

22.2.1.1 Addressability 747

22.2.1.2 Reagent exchange 749

22.2.2 Polymersomes and VirusLike Particles 751

22.2.3 Water-in-Oil Emulsion Droplets 752

22.2.3.1 Addressability 755

22.2.3.2 Reagent exchange 755

22.3 Microfabricated Chip Devices for Enzyme Compartmentalization and Screening 756

22.3.1 Microfluidic-Generated Emulsion Droplets 757

22.3.2 Microfabricated Arrays 762

22.3.2.1 Optical fiber microarrays 762

22.3.2.2 Elastomeric microarrays 763

22.3.2.3 Surface tension microarrays 765

22.4 Conclusion and Current Challenges 767

22.5 Future Improvements 769

23 Computational Enzyme Engineering：Activity Screening Using Quantum Chemistry&Martin R.Hediger 777

23.1 Motivation 778

23.2 Introduction 779

23.3 Methods 780

23.3.1 Calculation Engines 780

23.3.2 Molecular Modeling 782

23.3.3 Software 786

23.4 Applications 786

23.4.1 Overview 786

23.4.2 Engineering Candida antarctica Lipase B 787

23.4.3 Engineering Bacillus circulans Xylanase 793

23.5 Conclusions 800

24 In Silico Screening of Enzyme Variants by Molecular Dynamics Simulation&Hein J.Wijma 805

24.1 Potential Applications of MD Simulations For Improving Enzymes 805

24.2 Molecular Dynamics vs.Other in silico Methods 809

24.3 Improving Catalytic Activity by MD Screening 812

24.3.1 Transition-State Simulation 812

24.3.2 High-Energy Intermediate Simulation 814

24.3.3 Substrate Simulation with Near-Attack Conformations 815

24.3.4 Substrate Simulation with Monitoring of H Bonds 817

24.4 Predicting and Improving Binding Affiinity 818

24.5 MD Screening to Improve Enzyme Stability 819

24.6 Improving Correlation between MD and Experiment 822

24.6.1 Force Field Inaccuracies 822

24.6.2 Sampling Concerns 823

24.6.3 Other Concerns 824

24.7 Outlook and Further Possibilities 825

25 Kinetic Stability of Variant Enzymes&Jose M.Sanchez-Ruiz 835

25.1 Kinetics vs&Thermodynamics in Protein Stability 835

25.2 Mutation Effects on Kinetic Stability：A Description Based on the Transition State for Irreversible Denaturation 838

25.3 Kinetic Stability Linked to the Breakup of Interactions in the Transition State：Pro-dependent Proteases 841

25.4 Kinetic Stability Linked to Substantially Unfolded Transition States：Thioredoxin and Phytase Enzymes 842

25.5 Role of Solvation Barriers in Kinetic Stability：Lipases and Triose Phosphate Isomerases 848

25.6 Concluding Remarks 852

Index 859