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