© Oxford University Press. All Rights Reserved. BRIEF CONTENTS

Preface iii

1. Introduction to Enzymology 1 2. Mechanism of Enzyme Action 56 3. Enzyme Kinetics 89 4. Enzyme Inhibition 112 5. Regulation of Enzyme Activity 138 6. Coenzymes 162 7. Isozymes, Abzymes, Ribozymes, and Deoxyribozymes 222 8. Applications of Enzymes 254 9. Enzymes in Molecular Biology, Cloning, and Genetic Engineering 292 10. Enzyme Mutation 322 11. Synzymes 354 12. Enzyme Models—Artizymes 365 13. Immobilized Enzymes 374 14. Enzyme Technology and Enzyme Engineering 393

Lab Modules 435 Appendix 467 Further Reading 486 Index 487

Preface iii 1. Introduction to Enzymology 1 1.1 Biocatalysts 1 1.1.1 Evidence to Show that Enzymes are Proteins 2 1.1.2 Properties of Enzymes 2 1.1.3 Nomenclature and Classification of Enzymes 5 1.1.4 Factors Influencing Enzyme Activity 9 1.2 The Structure of Enzymes 12 1.3 Models for Monosubstrate Reaction 13 1.3.1 Fischer’s Lock and Key Model 13 1.3.2 Koshland’s Induced Fit Model 13 1.4 Models for Bisubstrate Reaction 14 1.4.1 Random Sequential Model 14 1.4.2 Ordered Sequential Model 15 1.4.3 Ping-pong Model 16 1.5 Monomeric Enzyme and Oligomeric Enzyme 16 1.6 Multisubstrate Reaction 18 1.7 Enzyme Localization—Histoenzymology 18 1.7.1 Definition of Histoenzymology 18 1.7.2 Applications 18 1.8 Enzyme Assay 20 1.8.1 Direct Assay 20 1.8.2 Two-step Coupled Assay or Linked Assay 20 1.8.2 Instrumental Techniques used in Enzyme Assay 22 1.9 Purification of Enzymes from Natural Resources 25 1.9.1 Need for Enzyme Purification 25 1.9.2 Steps in Enzyme Purification 26 1.10 Fractionation and Characterization of Proteins (Enzymes) 41 1.10.1 Analytical Ultracentrifugation 42 1.10.2 Gel Filtration Chromatography 43 1.10.3 Isoelectric Focusing 44 1.10.4 One-dimensional PAGE 44 1.10.5 Two-dimensional Gel Electrophoresis 45 1.10.6 HPLC/MS 46 1.10.7 Mass Spectrometry 46 1.10.8 Protein Identification by Peptide Mass Fingerprinting 48 2. Mechanism of Enzyme Action 56 2.1 Mechanistic and Intermediary Enzymology 56 2.1.1 Transition State and Activation Energy 56 2.1.2 Enzymes act as Catalyst by Lowering the Activation Energy 57

2.2 Strategies of Enzyme Action 59 2.2.1 Proximity Effect 59 2.2.2 Preferential Transition State Binding 60 2.2.3 Metal Ion Catalysis (Electrophilic Catalysis) 60 2.2.4 Covalent Catalysis (Nucleophilic Catalysis) 63 2.2.5 General Acid Base Catalysis 63 2.2.6 Concerted Acid Base Catalysis 65 2.3 Advantages of Mechanistic Enzymology 66 2.4 Multienzyme Complex 66 2.4.1 Types of Multienzyme Complex 66 2.4.2 Pyruvate Dehydrogenase Complex 68 2.4.3 Tryptophan Synthase Complex 69 2.4.4 Shikimate Pathway Complex or Arom Complex 69 2.4.5 DNA Polymerase Complex 69 2.5 Mechanism of Action of Some Select Enzymes 71 2.5.1 Lysozyme 71 2.5.2 Restriction Endonuclease 72 2.5.3 Oxidoreductases 73 2.5.4 NMP Kinase 76 2.5.5 Fructose-1,6-Bisphosphatase 77 2.5.6 Triose Phosphate Isomerase 78 2.5.7 Alkaline Phosphatase 78 2.5.8 Carbonic Anhydrase 79 2.5.9 Proteases 79 3. Enzyme Kinetics 89 3.1 Definition 89 3.2 Order of a Reaction 89 3.2.1 Zero-order Reaction 89 3.2.2 First-order Reaction 90 3.2.3 Second-order Reaction 90 3.2.4 Third-order Reaction 90 3.3 The Substrate Velocity Plot 91 3.3.1 Segments of Hyperbola 91 3.3.2 Michaelis-Menten Kinetic Postulates 92 3.4 The Michaelis-Menten Reaction 92 3.5 The MM Rate Equation 93 3.5.1 Parameters Involved in the Derivation of MM Rate Equation 93 3.5.2 Derivation 94 3.6 Constants in Enzyme-catalyzed Reaction 95 3.6.1 The Catalytic Constant 96 3.6.2 The Michaelis–Menten Binding Constant 96 3.7 Transformation of MM Plot Into Linear Plot 98 3.7.1 The Lineweaver-Burk Transformation 98 3.7.2 The Eadie-Hofstee Transformation 100 3.7.3 The Hanes Transformation 101

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3.8 Bisubstrate Reaction 102 3.8.1 Kinetics of Bisubstrate Reaction 102 3.8.2 Types of Bisubstrate Reaction 102 3.8.3 Reactions Involving a Ternary Complex 102 3.8.4 Formation of Ternary Complex by Two Models 103 3.8.5 Reactions Not Involving Ternary Complex 103 3.8.6 Equation for Bisubstrate Reaction 104 3.8.7 Significance of Vmax and Constants 105 3.9 Briggs-Haldane Equation 105 4. Enzyme Inhibition 112 4.1 Enzyme Inhibitors 112 4.2 Types of Enzyme Inhibition 112 4.2.1 Reversible Inhibition 113 4.2.2 Irreversible Inhibition 123 4.3 Active Site Mapping (ASM) 130 4.3.1 Definition 130 4.3.2 Mapping Agent 130 4.3.3 Protocol 130 4.3.4 Examples for Mapping 130 5. Regulation of Enzyme Activity 138 5.1 Need for Enzyme Regulation 138 5.2 Methods for Enzyme Regulation 138 5.2.1 Genetic Regulation 138 5.2.2 Covalent Modification 140 5.2.3 Proteolytic Cleavage (Zymogen Activation) 142 5.2.4 Allosteric Regulation 148 5.2.5 Regulation by Intracellular Compartmentalization 152 6. Coenzymes 162 6.1 Coenzymes and Cosubstrates 162 6.2 Functions and Types of Coenzymes 163 6.3 Vitamin-derived Coenzymes 163 6.3.1 Nicotinamide Coenzyme 163 6.3.2 Thiamine Pyrophosphate 170 6.3.3 Flavin Coenzymes 172 6.3.4 Coenzyme A 174 6.3.5 Pyridoxal Phosphate 177 6.3.6 Tetrahydrofolate 179 6.3.7 Cobamide Coenzymes 184 6.3.8 Biotin 189 6.3.9 Ascorbic Acid 191 6.3.10 Vitamin K 195 6.3.11 Lipoic Acid (Thicotic Acid) 198

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6.4 Non-vitamin-derived Coenzymes 199 6.4.1 Coenzyme Q10 200 6.4.2 Adenosine Triphosphate 203 6.4.3 Cytidine Triphosphate 206 6.4.4 Nucleotide Sugars 207 6.4.5 Glutathione 209 6.4.6 3¢-Phosphoadenosine-5¢-Phosphosulfate 210 6.4.7 S-Adenosyl Methionine 210 6.5 Other Coenzymes 212 6.5.1 Coenzyme B 212 6.5.2 Coenzyme F420 213 6.5.3 Coenzyme M 214 6.5.4 Tetrahydrobiopterin 214 6.6 Cofactors 215 6.6.1 Why Metal Ions are Good Cofactors? 215 6.6.2 Iron 215 6.6.3 Molybdenum 216 6.6.4 Copper 217 6.6.5 Zinc 218 6.6.6 Manganese 218 6.6.7 Calcium 218 6.6.8 Magnesium 219 6.6.9 Other Cofactors 219 7. Isozymes, Abzymes, Ribozymes, and Deoxyribozymes 222 7.1 Isozymes 222 7.1.1 Definition 222 7.1.2 Ontogenetic and Phylogenetic Variants 223 7.1.3 Homologous and Analogous Isozymes 223 7.1.4 Identification of Isozymes 223 7.1.5 Lactate Dehydrogenase 224 7.1.6 Isozymes of Hexokinase 227 7.1.7 Isozymes of Angiotensin-converting Enzyme 227 7.1.8 Creatine Phosphokinase Isoenzymes 228 7.1.9 Alkaline Phosphatase Isoenzymes 229 7.1.10 Applications of Isozymes 231 7.2 Abzymes 232 7.2.1 Definition 232 7.2.2 Sources of Abzymes 233 7.2.3 Protabzymes and DNA-Abzymes 233 7.2.4 Production of Abzymes 233 7.2.5 Examples for Abzymes 234 7.2.6 List of Reactions Catalyzed by Abzymes 237 7.2.7 Applications of Abzymes 237 7.2.8 Antibody-directed Abzyme Prodrug Therapy 238

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7.3 Ribozymes 238 7.3.1 RNA World Hypothesis 238 7.3.2 Ribozymes and Protein Enzymes 240 7.3.3 Types of Ribozymes 240 7.3.5 Reactions Catalyzed by Ribozymes 243 7.4 Deoxyribozyme 250 7.4.1 Definition 250 7.4.2 Reactions Catalyzed by DNAzyme 250 7.4.3 Advantages of DNAzymes 250 7.4.4 Disadvantages of DNAzymes 251 7.4.5 Applications of RNA Enzymes and DNA Enzymes 251 8. Applications of Enzymes 254 8.1 Enzymes as Diagnostic Agents 254 8.2 Enzymes as Analytical Agents 255 8.2.1 Coupled Assay 256 8.2.2 Estimation of Glucose 256 8.2.3 Estimation of Urea 256 8.2.4 Estimation of Uric Acid 257 8.2.5 Estimation of Cholesterol 257 8.3 Enzymes as Therapeutic Agents 258 8.4 Industrial Applications 258 8.4.1 Proteases 259 8.4.2 Carbohydrases 259 8.4.3 Lipase 261 8.4.4 Penicillin Acylase 262 8.4.5 Amino Acid Acylase 262 8.4.6 Cyclodextrin Glycosyl Transferase 262 8.4.7 Enzymes Remove Anti-nutritive Factors from Food 263 8.4.8 Enzymes as Debitterizing Agent 263 8.4.9 Enzymes as Aromatizer 264 8.4.10 Enzymes as Oxygen Scavenger 264 8.4.11 Microbiological Applications of Enzymes 264 8.5 Enzymes of Biological Importance 264 8.5.1 Angiotensin-converting Enzyme 265 8.5.2 Nucleotidase 269 8.5.3 Glucose-6-phosphate Dehydrogenase 270 8.5.4 Choline Esterase 273 8.5.5 Amylase 275 8.6 Enzyme Business in India and Abroad 277 8.6.1 Enzyme Standards 277 8.6.2 Enzyme Market 277 8.6.3 Enzyme Companies 278 8.6.4 Marketed Enzymes 279 8.6.5 Enzymes in Pipeline 288

Untitled-1 11 9/14/2010, 12:45 PM xii Contents 9. Enzymes in Molecular Biology, Cloning, and Genetic Engineering 292 9.1 Enzymes in Replication 292 9.1.1 DNA Polymerase 293 9.1.2 DNA Gyrase 296 9.1.3 Helicase 297 9.1.4 Primase 297 9.2 Enzymes in Transcription 297 9.2.1 Components Required for Transcription 298 9.2.2 RNA Polymerase Activity 298 9.2.3 Poly Adenylyl Polymerase 300 9.2.4 Ribonuclease P and Ribonuclease III 300 9.3 Enzymes in Translation 301 9.3.1 Components Involved in Translation 301 9.3.2 Aminoacyl tRNA Synthetase 302 9.3.3 Transformylase 302 9.3.4 Peptidyl Transferase 303 9.3.5 Deformylase 303 9.4 Nucleases 303 9.4.1 Restriction Endonucleases 304 9.4.2 Deoxyribonuclease 307 9.4.3 S1 Nuclease 308 9.4.4 Ribonuclease 309 9.4.5 Exonuclease VII 310 9.4.6 Mung Bean Nuclease 311 9.5 Ligase 311 9.5.1 Action of Ligase 311 9.5.2 Types of Ligase 311 9.5.3 Uses of Ligase 311 9.6 Alkaline Phosphatase and Polynucleotide Kinase 312 9.7 Proteinase K 312 9.7.1 Activity and Peculiarity 312 9.7.2 Uses of Proteinase K 312 9.8 Terminal Deoxynucleotidyl Transferase 313 9.8.1 Functions 313 9.8.2 Applications 313 9.9 Reverse Transcriptase 313 9.9.1 Examples 313 9.9.2 Activities of Reverse Transcriptase 314 9.9.3 Functions 314 9.9.4 Applications 314 9.10 Telomere and Telomerase 315 9.11 Reporter Enzymes 315 9.11.1 Examples 315 9.12 Other Enzymes 316 9.12.1 EcoRI Methylase 316 9.12.2 Sequenase 316

Untitled-1 12 9/14/2010, 12:45 PM Contents xiii 10. Enzyme Mutation 322 10.1 Definition 322 10.2 Subtilisin 322 10.3 Types of Mutation 323 10.3.1 Site-direected Mutagenesis 323 10.3.2 Random Mutagenesis 326 10.3.3 Cassette Mutagenesis 336 10.3.4 Directed Mutagenesis 336 10.3.5 Stretch Deletion 336 10.3.6 Transposon Mutagenesis 337 10.4 Selection of Mutants 337 10.4.1 Filamentous Gene M13–A Classic Selection Vehicle 337 10.4.2 Pros and Cons of M13 Bacteriophage 338 10.5 Common Mutations in Target Amino Acid Residues of Enzymes 339 10.5.1 Simple Deletion Mutation 340 10.5.2 Multipart Deletion 341 10.5.3 Oxy Deletion, Hydroxy Deletion, and Sulpho Deletion 343 10.5.4 Histidine Open Mutation 343 10.5.5 Substitution Mutation 344 10.6 Role of Mutation in Understanding the Catalytic Process 345 10.6.1 Activation of Tyrosine 345 10.6.2 Charging of tRNA by Tyrosine 345 10.6.3 Threonine-40 and Histidine-45 Play the Binding Role 345 10.6.4 Cysteine-35 and Histidine-48 Play the Catalytic Role 347 10.6.5 Lysine-230 and Lysine-233 Catalyze the Induced Fit Process 347 10.6.6 Thr Pro Mutation Enhances the Rate Activation of Tyrosine 347 10.6.7 Stretch Deletion Reveals the Structural Features 347 10.7 Enzyme Mutations in Diseased State 347 10.7.1 Significance of Mutated Ferrochelatase in Erythropoietic Protoporphyria 348 10.7.2 Mutated 11-cis-Retinol Dehydrogenase and Fundus Albipunctatus 348 10.8 Examples of Reactions Catalyzed by Mutated Enzymes 348 11. Synzymes 354 11.1 Definition of Synzymes 354 11.2 Criteria for Creating Synzymes 354 11.3 Flavopapain – A Typical Example for Synzyme 355 11.4 Some Important Synzymes 355 11.4.1 Synthetic Protease from Met-myoglobin 356 11.4.2 Esterase from Polyglutamate 356 11.4.3 Chymotrypsin from Amines 356 11.4.4 Transaminase from Cyclodextrins 358 11.4.5 Synthetic Ascorbic Oxidase from Myoglobin 358 11.5 Antioxidant Synzymes in Blood Substitute 359 11.5.1 Blood Substitute 359 11.5.2 Advantages of Blood Substitutes over Human Red Cells 359 11.5.3 Polymerized Hemoglobin Linked to Antioxidant Enzymes 360 11.5.4 Liposomal Encapsulated Hemoglobin 360

Untitled-8 13 9/15/2010, 11:34 AM xiv Contents 12. Enzyme Models–Artizymes 365 12.1 Definition 365 12.2 Synonyms for Enzyme Models 365 12.3 Biomimetics 366 12.4 Features of Enzyme Models 366 12.5 Advantages of Enzyme Models 367 12.6 Tailoring Enzyme Models 367 12.6.1 Specific Catalytic Groups used for Artizymes 367 12.6.2 Specific Substrate 368 12.6.3 Propinquity Matrix 368 12.7 Demerits of Artizymology 368 12.8 Examples of Engineered Artizymes 368 12.9 Artificial Metallozymes 369 12.9.1 Antibody-rhodium Catalyst 369 12.9.2 Enantioselective Organometal Catalyst 370 12.10 Application of Artizymes 370 12.10.1 Catalytic Efficiency of Cytochrome Oxidase 370 13. Immobilized Enzymes 374 13.1 Definition 374 13.2 Advantages of Immobilization 374 13.3 Methods of Immobilization 375 13.3.1 Entrapment Method 375 13.3.2 Carrier Binding Method 379 13.3.3 Crosslinking Method 384 13.3.4 Nanocrystalline Method 386 13.4 Examples for Immobilized Enzymes 386 13.4.1 Immobilized Glucosidase and Xylose Isomerase 387 13.4.2 Immobilized L-Aminoacylase 387 13.4.3 Immobilization of Microbial Cells 388 13.5 Applications of Immobilized Enzymes 389 14. Enzyme Technology and Enzyme Engineering 393 14.1 Production of Enzymes of Interest 393 14.1.1 Microbial Production of Enzymes 394 14.1.2 Fermentation in Bioreactor 394 14.1.3 Components of a Bioreactor 394 14.1.4 Enzyme Production by Microbial Culture 395 14.2 Enzyme Reactor 396 14.2.1 Components of an Enzyme Reactor 396 14.2.2 Types of Enzyme Reactor 396 14.2.3 Applications of Enzyme Reactor 399 14.2.4 Immobilized Heparinase Reactor Design 402 14.2.5 Immobilized Invertase Reactor 403

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14.3 Biosensors and Biochips 404 14.3.1 Biosensors 404 14.3.2 Biochips 407 14.4 Enzyme Electrode 409 14.4.1 Principle and Components 409 14.4.2 Mediators 409 14.5 Inhibited Enzyme Electrode 410 14.6 ELISA 410 14.6.1 Principle of ELISA 410 14.6.2 Steps in ELISA 412 14.6.3 Enzymes Used in ELISA 412 14.7 Engineered Enzymes 413 14.7.1 Extremophiles 413 14.7.2 Types of Extremophiles and Their Habitats 413 14.7.3 Extremozymes 414 14.7.4 Engineered Extremozymes 414 14.7.5 Applications of Extremophiles/Extremozymes 417 14.7.6 Factors Stabilizing Extremozymes 419 14.7.7 Hyperthermophiles 419 14.7.8 Psychrophiles 424 14.7.9 Halophiles 424 14.7.10 Acidophiles 425 14.7.11 Alkaliphiles 426 14.7.12 Additional Extremophiles 426 14.8 Enzymes and Bioinformatics 427 Lab Modules 435

LM1 Extraction of Enzymes from Plant Sources and Assay of Enzyme Activity 437 LM2 Extraction of Enzymes from Animal/Human Sources and Assay of Enzyme Activity 441 LM3 Determination of Optimum Reaction Conditions for Enzyme Activity 450 LM4 Enzyme Linetics—Michaelis–Menten Parameters 455 LM5 Enzyme Immobilization 458 LM6 Fractionation of Proteins by Ammonium Sulphate and Desalting 460 LM7 Determining the Molecular Weight of Proteins by SDS-PAGE 462 Appendix 467 Glossary of Terms 469 Hematological Terms 471 Biochemical Reference Values 473 Atomic Number and Atomic Weight of Elements 474 Standard Reduction Potentials of Some Oxidation–Reduction Half Reactions 475 Properties of Acids 475 Pi Values of Amino acids 476 Preparation of Common Lab Solutions 476 Molecular Weight of Proteins 476

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Measures of Important Parameters 477 Specific Rotation of Some Carbohydrates 478 Units for Radioactivity 478 Half-Life of Radioisotopes 478 Filters for Colorimetry 478 Properties of Common Oils 478 Absorption Maxima and Absorption Coefficient of Some Biomolecules 479 Gels and Stains Used in Electrophoresis 479 Electrophoretic Pattern of Serum Proteins 480 List of Chromatography Matrices 480 Preparation of Buffers 481 Preparation of Buffered Saline 484 Further Reading 486 Index 487

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INTRODUCTION TO

James Batcheller Sumner ENZYMOLOGY was the first scientist to crystallize enzymes.

LEARNING OBJECTIVES

After reading this chapter, the reader would be able to understand and appreciate the following: ∑ Definition for enzyme and terminologies involved in enzymology ∑ Nomenclature and classification of enzymes ∑ Influence of substrate concentration, temperature and pH on enzyme activity ∑ Models for monosubstrate and bisubstrate reactions ∑ Histoenzymology and enzyme assay ∑ Techniques used in enzyme assays, such as spectrophotometry, fluorimetry, luminometry, manometry, enthalpimetry, and radiometry ∑ Steps and protocols involved in the purification of enzymes from natural resources ∑ Methods used for fractionation and characterization of proteins (enzymes)

1.1 BIOCATALYSTS

Enzymes are a colloidal, high molecular weight, non-dialyzable, denaturable, structur- ally diverse group of proteins. Biochemical reactions taking place within the living cells are catalyzed by enzymes (hence the name, biocatalysts). Enzymes increase the rate of reaction without undergoing any change themselves and without altering the equilib- rium of the reaction. Like any other protein, an enzyme is an amino acid polymer with added cofactors and other post-translational modifications. Often, most of the amino acid polymers are indirectly involved with the enzyme function, perhaps providing ancillary structure or connectivity, indirect activity regulation, or molecular identification of the enzyme. As a result, most enzymes are large molecules weighing many kilo Daltons (kDa). Enzymes catalyze metabolic reactions, by which ∑ Nutrients are degraded ∑ Chemical energy is conserved and transformed

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∑ Biomolecules are synthesized from their precursors ∑ Cellular activity is regulated in response to various metabolic signals

1.1.1 EVIDENCE TO SHOW THAT ENZYMES ARE PROTEINS After crystallizing urease, James Sumner discovered that enzymes are purely proteins (ribozyme is an exception). Enzymes share the following properties with proteins: ∑ The proportion of carbon, hydrogen, nitrogen, and sulphur in enzymes are similar to protein. ∑ Like proteins, enzymes act as ampholytes ∑ Enzymes are susceptible to denaturation with a consequent loss of activity ∑ Enzymes can elicit specific antibodies on being injected into an animal body

Enzyme units The actual molar amount of the enzyme cannot be determined and therefore, it is unknown. However, the amount can be expressed in terms of its activity. There are four ways of expressing enzyme activity. International Unit (IU) One IU of an enzyme is the amount of enzyme that catalyzes the formation of one micromole of product in one minute under optimal conditions of pH, temperature, and ionic strength. Katal One Katal is the amount of enzyme catalyzing the conversion of one mole of substrate to product in one second. 1 Katal = 6 ¥ 107 IU.

Turnover number (kcat ) In enzymology, the turnover number (also termed kcat) is defined as the maximum number of moles of substrate that an enzyme can convert to product per catalytic site per unit time. It is also called molecular activity. For example, carbonic anhydrase has a turnover number of 400,000 s–1, which means that each car-

bonic anhydrase molecule can produce up to 400,000 molecules of product (CO2) per second. Specific activity (U/mg) Specific activity is the number of units of enzyme activity per mil- ligram of total protein present. Enzyme concentration [E] is units/mg protein ¥ mg protein/ ml.

1.1.2 PROPERTIES OF ENZYMES Enzymes possess the following distinctive properties: ∑ Catalytic power ∑ Specificity ∑ Regulatory power ∑ Milder reaction conditions

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· Reversibility · Colloidal nature · Denaturation

Catalytic power Catalytic power is also called as the catalytic efficiency or the rate enhancement power. The remarkable property of the cells to carry out reactions at low temperature, pressure and in dilute aqueous medium is obviously due to the catalytic power of the enzymes. An enzyme increases the reaction rate by 106 to 1012 times over an uncatalyzed reaction. The ratio of the rate of catalyzed reaction to the rate of uncatalyzed reaction is called the catalytic power of an enzyme. The catalytic power of some enzymes is shown in Table 1.1. Turnover numbers of selected enzymes are shown in Table 1.2. (See Chapter 2 for the energy level diagram.)

Table 1.1 Catalytic power of some enzymes Enzymes Uncatalyzed rate Catalyzed rate Catalytic power

(Kuncat/s)(Kcat/s)(Kuncat /s : Kcat /s)

AMP nucleotidase 1.0 ´ 10–11 60 6 ´ 10–11 Carboxy peptidase A 3.0 ´ 10–9 578 1.9 ´ 10–11 Triose phosphate isomerase 4.3 ´ 10–6 4300 1 ´ 10–11 Carbonic anhydrase 1.3 ´ 10–1 1 ´ 106 7.7 ´ 10–11 Urease 3 ´ 10–10 3 ´ 104 1014

Specificity Specificity refers to the extraordinary ability of the enzyme to recognize a specific substrate to catalyze a specific reaction. Enzymes exhibit six types of specificity:

Absolute specificity Specificity of an enzyme towards a single substrate is called absolute specificity. For example, urease is specific to urea. Urea Urease Carbon dioxide + Ammonia

Dual specificity The recognition of two different substrates by an enzyme is called dual specificity. For example, sucrase acts on sucrose and raffinose. Sucrase Sucrose Glucose + Fructose Sucrase Raffinose Fructose + Mellibiose

Group specificity Specificity of an enzyme towards a group of closely related compounds is called group specificity. For example, alcohol dehydrogenase (ADH) acts on ethanol and other higher alcohols.

Table 1.2 Turnover number of selected Ethanol + NAD+ ADH Acetaldehyde + NADH + H+ enzymes Propanol + NAD+ ADH Propanaldehyde + NADH + H+ Enzymes Turnover number Geometrical specificity Recognition of cis-trans iso- Carbonic anhydrase 6 ,000,000 mers by enzymes is called geometrical specificity. For Catalase 93,000 example, fumarase acts on fumarate (cis-form) and Acetylcholine esterase 25,000 malate (trans-form). Lactate dehydrogenase 1,000 Fumarate (cis-form) Fumarase Malate (trans-form) RNAse 800 Optical specificity Specificity of enzymes towards b-galactosidase 200 chiral substances (D&L isomers) is called optical DNA polymerase 15 specificity. For example, racemase acts on D and L Tryptophan synthetase 2 isomers.

Lysozyme 0.5 Ala racemase D-Alanine L-Alanine

Bond specificity Selectivity of enzymes to a specific type of bond in a compound for subsequent hydrolysis is called bond specificity. For example, glycosidase cleaves glycosidic bond, peptidase (pepsin, trypsin) cleaves peptide bond, and esterase cleaves ester bond.

Chymotrypsinogen Trypsin Chymotrypsin (inactive)Pepide bond cleavage (active)

Regulatory power Another important property of an enzyme is its susceptibility to regulation, i.e. the catalytic power is controllable depending on the metabolic needs of the cell. Enzymes can be regulated by several mechanisms—covalent modification, post-translational modification, proteolytic cleavage, and genetic control. (All these methods are discussed in Chapter 5).

Milder reaction conditions Enzyme catalysis occurs under milder conditions of temperature, atmospheric pressure, and nearly neutral pH, except for extremozymes, which are active under extreme conditions (see Chapter 14.) (A chemically catalyzed reaction, in vitro, requires extremes of temperature, pressure, and pH)

Reversibility Enzymes can catalyze reactions in both directions depending upon the availability of a suitable energy source and suitable solvent conditions.

Cha 1.pmd 4 9/9/2010, 6:54 PM Introduction to Enzymology 5 Colloidal nature Enzymes, being high molecular weight proteins, exhibit colloidal properties, due to which they present a large surface area for the reaction to take place.

Denaturation Enzyme denaturation is commonly defined as any noncovalent change in its structure. This change may alter the secondary, tertiary, or quaternary structure of the enzyme molecules. Enzymes are thermolabile and pH sensitive. Hence, they are denatured by high temperature, strong acids, and strong alkali. When proteins are exposed to increasing temperature (except hyperthermozyme—Chapter 14), interactions responsible for tertiary structure are broken. A further rise in temperature results in breaking of the hydrogen bond, increasing the flexibility of the protein, thus leading to denaturation. When enzymes are exposed to very high pH at elevated temperatures, it results in alkaline hydrolysis of peptide bonds. Proteins containing acid labile groups irreversibly lose their function even with mild acid treatment. This is due to the breaking of specific covalent bonds. Exposure to strong acid at elevated temperatures will first release amide nitrogen from glutamine and asparagine groups, and eventually lead to the hydrolysis of peptide bonds.

1.1.3 NOMENCLATURE AND CLASSIFICATION OF ENZYMES Enzymes are generally named by adding the suffix—‘ase’ to the substrate of the reaction. For example urease. Other enzymes acquired names bearing little resemblance to their activity, such as the peroxide-decomposing enzyme catalase, or the proteolytic enzymes (proteases) of the digestive tract, trypsin and pepsin, or the hydrolytic enzyme, hydrolase.

Major classification According to the International Enzyme Commission constituted by the International Union of Biochemistry (1956), enzymes are divided into six major classes depending on the type of reaction catalyzed. Major classification of enzymes is shown in Table 1.3.

Subclass and sub-subclass Each major class is further divided into subclass and each subclass is redivided into subsubclass. Subclasses are shown in Table 1.4.

Cha 1.pmd 5 9/9/2010, 6:54 PM 6 Enzymology red + B – Oxd A – B A – H + B OH = X – Y = X – A + B ´ ´ + A + B – C AY – BX AY A enzyme ´ O A ´ ´ ´ 2 oxd ´ + + B Model reaction catalyzed Model reaction + B + ADP + Pi ADP + Red – A – B + C A A A Example Alcohol dehydrogenase Phosphate synthetase Phosphate Cyt. oxidase Transaminase Major classification of enzymes Table 1.3 Table Name of the reaction catalyzedreaction by the specific class of Non-hydrolytic bondNon-hydrolytic of double bonds Histidase Aldolase, X – BY A rearrangement of groupsisomerase Phosphohexose ATP* of expense two substrates Oxidoreductase Redox Lyase (Desmolase) cleavage and generation Isomerase Intramolecularisomerasephosphate Triose X – BY A Ligase(Synthetase) theat formation Bond Carbamoylsynthetase, Glutamine ATP + B A Transferasebetweentransfer Group Hydrolase Hexoseaminase Hydrolytic bond cleavage Alkaline phosphotase, Urease – A B + H Class An enzyme that catalyzes bond formation without the expense of ATP is called synthase ATP An enzyme that catalyzes bond formation without the expense of *

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Table 1.4 Subclasses of enzymes Class Subclass Example Reaction catalyzed

Oxidoreductase Dehydrogenase Lactate dehydrogenase Removal of 2 hydrogen atoms Malate dehydrogenase with double bond formation Oxidase Cytochrome oxidase Reduction of oxygen

Peroxidase Catalase Reduction of H2O2 Hydroxylase Phenylalanine-4- Introduction of OH groups hydroxylase Oxygenase Tryptophan oxygenase Incorporation of molecular oxygen Oxidative deaminase Amino acid oxidase Oxidation of amino acid with the liberation of NH3 Transferase One carbon transferase Methyl transferase Transfer of one-carbon group Hydroxy methyl transferase Formyl transferase Aldehyde and ketone Acetaldehyde Transfer of aldehyde/keto group transferase transferase Acyl transferase Aminoacyl transferase Transfer of acyl/acetyl group to a suitable acceptor Glycosyl transferase Hexosyl transferase Transfer of glycosyl group

Alkyl transferase Ethyl transferase Transfer of alkyl other than methyl

N-transferase Amino transferase Transfer of nitrogenous group Phospho transferase Kinase Transfer of phosphoryl group

Sulpho transferase Dehydro epiandroster- Transfer of sulphur containing one Sulphotransferase group Choline sulphokinase Hydrolase Esterase Acetylcholine esterase Hydrolysis of ester Peptidase Trypsin Hydrolysis of peptide bond Glycosidase Lysozyme Hydrolysis of glycosidic bond

(Contd.)

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Table 1.4 (Contd.) Class Subclass Example Reaction catalyzed

Phosphatase ALP Hydrolysis of phosphoric acid ester Deaminase Glucosamine-6-P- Hydrolysis of amines deaminase, Glutaminase Deamidase Nicotinamide deamidase Hydrolysis of amides Lyase C = C lyase Aldolase Cleavage of carbon-carbon bond C = O lyase DNA lyase Cleavage of carbon-oxygen bond C = N lyase Ammonia lyase Cleavage of carbon- nitrogen bond

Isomerase Racemase Alanine racemase Interconversion of optical isomers Epimerase Maltose epimerase Interconversion of epimers Cis-trans Isomerase Fumarase Interconversion of geometrical isomers Ligase C = O ligase Tyrosine-RNA ligase Formation of C – O bond C = S ligase Acetate CoA ligase Formation of C – S bond C = N ligase NAD+ synthase Formation of C – N bond Tryptophan synthase C = C ligase Pyruvate carboxylase Formation of C – C bond

Enzyme commission code (EC) Each enzyme is assigned a code by the enzyme commission. The code number is called the enzyme code. It has four digits. The first integer represents the major class, the second integer represents the subclass, the third integer represents the sub-subclass, while the fourth integer represents the individual serial number of that enzyme in the sub-subclass. Thus, a series of four numbers serves to specify a particular enzyme. To illustrate, consider the enzyme that catalyzes this reaction:

D-Glucose-6-phospho transferase ATP + D-Glucose ADP + D-Glucose-6-phosphate

Class In the above reaction, a phosphate group is transferred from ATP to the – OH group of the sixth carbon of glucose, so the enzyme is a transferase (Class 2).

Subclass Enzymes transferring phosphorus-containing groups are called phosphotransferases which comes under seventh category of transferase class, so the subclass is 7.

Sub-subclass If an alcohol group is the acceptor of the phosphate group, it refers to the first category of the phosphotransferases. Therefore, the subsubclass is 1. Entry 2 in this sub-subclass is ATP. Therefore, the EC number for D-glucose-6-phosphotransferase is 2.7.1.2.

1.1.4 FACTORS INFLUENCING ENZYME ACTIVITY Various factors influence the activity of enzymes. These factors include substrate concentration, reaction temperature, pH of the buffer or reaction medium, oxidation, and radiation exposure. The effect of these factors on the rate of enzyme catalyzed reaction is discussed below.

Effect of substrate concentration The effect of substrate concentration on enzyme activity can be described by plotting velocity (V) against substrate concentration [s], as shown in Fig. 1.1. The plot is a rect- angular hyperbola with two distinct regions: (a) linear region, and (b) plateau region.

Transition point

Vmax Plateau [ZOKSE ; FOK ] Linear region [FOK ] V S ZOKS = Zero-order kinetics with respect to substrate Vmax FOKE = First-order kinetics with respect to enzyme 2 concentration FOKS = First-order kinetics with respect to substrate concentration Km

[S]

Fig. 1.1 Effect of substrate concentration on enzyme activity

Linear region At the initial stage of the reaction the velocity of the enzyme catalyzed reaction increases linearly with an increase in substrate concentration (shown as the linear region). This is also termed initial velocity of the reaction. At this stage, the substrate concentration is low and the reaction follows first order kinetics with respect to substrate concentration. Generally, it is assumed that the velocity of the enzyme-catalyzed reaction is directly proportional to the enzyme concentration in a reaction mixture.

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Plateau region As the substrate concentration is raised gradually, the velocity reaches a maximum (Vmax). After Vmax the velocity is constant even with an increase in substrate concentration. This leads to a plateau region. At this region, the reaction rate follows zero order kinetics with respect to substrate, but first order kinetics with respect to enzyme concentration. The point in the graph, where the linear plot is transformed into a plateau is called the saturation point. At this point the active site of the enzyme is completely saturated

with the substrate. Substrate concentration at which the rate is Vmax/2, i.e. the rate is half of the maximal velocity is called km.

Effect of temperature The effect of temperature on the rate of the enzyme catalyzed reaction is represented as plot of velocity versus. temperature. This is shown in Fig. 1.2. The plot is bell shaped with three regions: ∑ ascending segment, ∑ peak, and ∑ descending segment.

Vmax Peak

Ascending limb V ( in collision) Descending segment (active site disruption) Optimum (threshold) temperature T °C

Fig. 1.2 The effect of temperature on the rate of an enzyme-catalyzed reaction

The rate of enzyme-catalyzed reaction increases with the initial rise in temperature in accordance with the Arhenius equation: –DG*/RT K = Ae where, K is the kinetic rate constant for the reaction, A is the Arrhenius constant, also known as the frequency factor, DG* is the standard free energy of activation (kJ M –1), which depends on entropic and enthalpic factors, R is the gas law constant, and T is the absolute temperature.

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The initial rise in temperature increases the probability of effective collision between the reactive groups due to increase in kinetic energy, and hence the velocity increases linearly (shown as an ascending segment). The velocity ‘V’ is enhanced for every 10°C

is called temperature coefficient, denoted as Q10. But after a certain limit of temperature, called the optimum temperature, the velocity reaches a maximum (peak), then decreases gradually (the descending segment). The descending segment represents that beyond the optimum temperature there is a fall in velocity. It may be due to covalent changes such as the deamination of asparagine residues, or noncovalent changes, such as the rearrangement of the protein chain, or inactivation by heat denaturation.

Effect of pH The effect of pH on the velocity of an enzyme-catalyzed reaction is represented in Fig. 1.3.

Vmax Ascending segment ( inE+Sbinding)

V Descending segment (E–S dissociation)

Optimum pH

Fig. 1.3 The effect of pH on the velocity of an enzyme-catalyzed reaction

The bell-shaped curve of pH versus V is similar to that of T versus V. In the initial stage of the reaction the velocity increases with the increase in pH due to increase in enzyme-substrate binding. At a particular pH, called the optimum pH (usually 6–8) the velocity reaches a maximum. Beyond the optimum pH the 3-D structure of the enzyme is altered, leading to a dissociation of E–S complex and a fall in velocity. As the optimum pH is between 6–8 most of the enzymes display a bell-shaped curve, but pepsin is an exception. The optimum pH of pepsin is 2 (which exists in acidic gastric juice for digesting proteins), and the curve, is shown in Fig. 1.4.

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Half bell-shaped curve V

Fig. 1.4 The effect of pH on the velocity of pepsin

Effect of oxidation Oxidation of the sulphydryl group (–SH) in the active site by the oxidizing agent leads to disulphide bridging (S–S), resulting in loss of enzyme activity.

Effect of radiation Exposure to high energy (short wavelength) radiations like X-rays, b-rays and g -rays, leads to conformational change and loss of enzyme activity. UV rays also inactivate enzymes.

1.2 THE STRUCTURE OF ENZYMES

Enzymes have properly folded active conformation. Part of the enzyme responsible for catalysis is called active site. Active site is functionally divided into two subsites. Specificity subsite (substrate binding site) is the part of the active site where recognition of the substrate takes place. Reaction subsite is the part of the active site where chemistry occurs. A scheme of enzyme-substrate binding leading to catalysis is shown in Fig. 1.5. Electrostatic forces and torques can steer the substrate (negatively charged) into its binding site on the protein. Some binding sites are normally shielded from the solvent and can be kept closed by salt links between groups on the protein surface. If the correct substrate disrupts these salt links it can gain access to the binding site. This is known as gated binding. Electrostatic interactions, particularly salt links and hydrogen bonds between the ligand and the protein can contribute to the affinity and specificity of binding and to the orientation of the ligand in the binding site. Substrate (S) binds to the active site of the enzyme (E) forming enzyme-substate complex. An enzyme-substate complex is converted into a high-energy state called transition state complex (ES‡). Subsequently, the transition state is converted into product (P) and the free enzyme (E) is released.

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E + S ES ES‡ P + E – – – – –

+ + + + + + – – + + – – + – +

(a) (b) (c)

Fig. 1.5 Enzyme-substrate binding steered by electrostatic forces—gated binding

1.3 MODELS FOR MONOSUBSTRATE REACTION Monosubstrate reaction is given as: E + S ES P + E A monosubstrate reaction is explained by two models: ∑ Fischer’s lock and key model, and ∑ Koshland’s induced fit model.

1.3.1 FISCHER’S LOCK AND KEY MODEL According to this model the active site of the enzyme is rigid and predetermined to be complementary to the substrate. Thus, the substrate fits into the active site as the key fits into the lock. This model is shown in Fig. 1.6.

S+E

(Enzyme) (Substrate) (E–S complex)

Fig. 1.6 Fischer’s lock and key model for a monosubstrate reaction

1.3.2 KOSHLAND’S INDUCED FIT MODEL In this model, the active site of the enzyme is flexible and changes shape on substrate binding, i.e. the complementarity is induced after the binding of the substrate to the enzyme. This model is shown in Fig. 1.7.

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(E) (S) (E–S complex)

Fig. 1.7 Koshland's induced fit model for monosubstrate reaction

1.4 MODELS FOR BISUBSTRATE REACTION A bisubstrate reaction is represented as A + B E P + Q A bisubstrate reaction is described by three models. All the models are explained by the Cleland diagram or Cleland notation.

A B P Q

AEB PEQ QE E A & B = Substrates E P & Q = Products BAE QEP PE E

B A Q P (a)

Creatine kinase (CK) ATP + Creatine ADP + Creatine phosphate (Cr) (CrP) ATP Cr ADP CrP

ATP CK Cr ADP CK CrPECrP E CK Cr CK ATP CrP CK ADP E ADP E

Cr ATP CrP ADP (b)

Fig. 1.8 (a) Cleland notation for random sequential model (Random single displacement model) and (b) Cleland notation for creatine kinase

1.4.1 RANDOM SEQUENTIAL MODEL Ternary complexes are formed in this model. Creatine kinase is the best example for this model. It is also known as random single-displacement model.

Cha 1.pmd 14 9/9/2010, 6:54 PM Introduction to Enzymology 15 The Cleland notation for this model along with the example, creatine kinase, is shown in Figs 1.8 (a) and (b). ∑ Substrate (A/B) binds to enzyme (E) in random order ∑ Enzyme-substrate ternary complex is formed (AEB/BEA) ∑ Enzyme-substrate ternary complex is converted into enzyme-product ternary complex (PEQ/QEP) ∑ The products are released one-by-one at random (Q+P/P+Q) ∑ Free enzyme is regenerated (E)

1.4.2 ORDERED SEQUENTIAL MODEL Alcohol dehydrogenase is the best example for this model. The Cleland notation for this model is depicted in Figs 1.9 (a) and (b). It is also known as ordered single-displacement model.

A & B = Substrates P & Q = Products A B Q P

E AEB PEQ PE E

Fig. 1.9 (a) Cleland notation for ordered sequential model

(NAD+ ) (Ethanol) ADH NAD+ ADH NAD+ ADH Ethanol NADH ADH (Aldehyde)

Aldehyde

NADH— ADH

ADH

(NADH) Fig. 1.9 (b) Cleland notation for alcohol dehydrogenase (Here the cofactor NAD is considered as one of the two substrates.)

∑ The substrate binds to the enzyme (E) in a definite order. ∑ The leading substrate is A (obligatory substrate or compulsory substrate). ∑ The trailing substrate is B. ∑ The enzyme-substrate ternary complex (AEB) is formed. ∑ AEB is converted into enzyme-product ternary complex (PEQ).

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∑ The products are released in definite order (Q is released first, followed by P). ∑ Free enzyme is regenerated (E).

1.4.3 PING-PONG MODEL A binary complex is formed in this model. Transaminase is the best example for this model. In this model, one of the substrates should be converted into the product before the next substrate binds to the enzyme. Cleland notation for this model is shown in Figs 1.10 (a) and (b). In this model, one of the substrates should be converted into the product before the next substrate binds to the enzyme. Transaminase Glutamate + Oxaloacetate a-ketoglutarate + Aspartate (Glu) (OA) (a KG) (Asp)

A PB Q

EEAEPE EB EQ E (a)

Glu αKG OA Asp

TA Glu TA αKG TA TA TA OA TA Asp TA (b)

Fig. 1.10 Cleland notations for (a) ping-pong model and (b) for Transaminase

In this model, one of the substrate should be converted into product before the next substrate binds to the enzyme. ∑ The leading substrate (A) binds to the enzyme (E). ∑ Enzyme-substrate binary complex is formed (AE). ∑ Enzyme-product binary complex is formed (PE). ∑ The product (P) is released with the regeneration of free enzyme. ∑ The trailing substrate (B) binds to enzyme (E). ∑ Enzyme-substrate binary complex is formed (BE). ∑ Enzyme-product binary complex is formed (QE). ∑ The product (Q) is released. ∑ Free enzyme is regenerated (E).

1.5 MONOMERIC ENZYME AND OLIGOMERIC ENZYME

Enzymes with single non-dissociable polypeptide chain are called monomeric enzymes. They are usually composed of 100 to 300 amino acids and mostly synthesized as

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Table 1.5 Members of monomeric enzymes zymogens and their activities regulated by proteolytic Enzyme type Examples cleavage (see Chapters 3 and 5). Most of the known monomeric enzymes come under the class of hy- Protease Chymotrypsin drolases, including peptidase, protease (serine pro- Trypsin teases, thiol proteases), nuclease, and glycosidase. Elastase Some of the monomeric enzymes are enlisted in Thrombin Table 1.5. Subtilisin Enzymes with two or more polypeptide chains Pepsin are called oligomeric enzymes. Their component Ficin polypeptide chains are called subunits. Examples for Carboxypeptidase A oligomeric enzymes and their subunits are shown Carboxypeptidase B in Table 1.6. Subunits of an oligomeric enzyme may be identical or nonidentical. Identical subunits are Nuclease Ribonuclease called protomers. Enzymes with two subunits are Glycosidase Lysozyme called dimers and those with three and four subunits are called trimers and tetramers, respectively. Oli- gomeric enzymes have higher molecular weight than monomeric enzymes. These enzymes are regulated by feed back inhibition or allosteric regulation (see Chapter 5). Examples for oligomeric enzymes include lactose synthase, lactate dehydrogenase, and pyruvate dehydrogenase. Lactose synthase is a dimer. One subunit is inactive and is called a-lactalbumin and the other one is called N-acetyl lactosamine synthase, which catalyze the following reaction in carbohydrate metabolism: UDP-galactose + N-acetyl glucosamine ↔ UDP + N-acetyl lactosamine In the presence of a-lactalbumin, N-acetyl lactosamine synthase catalyze the synthesis of lactose as follows. This reaction is important in a lactating mother: UDP-galactose + Glucose ↔ UDP + Lactose Lactate dehydrogenase is a tetramer and each subunit catalyses the same reaction: Lactate + NAD+ ↔ Pyruvate + NADH + H+ The enzyme subunits exist in two forms based on the difference in amino acid composition. They are M form, which predomi- Members of oligomeric enzymes Table 1.6 nates in skeletal muscle, and H form, which Oligomeric enzyme Number of subunits predominates in the heart. These forms are present called isozymes (see Chapter 7). Pyruvate dehydrogenase is a multienzyme Lactate dehydrogenase Four complex, which catalyzes the synthesis of the Lactose synthase Two two-carbon unit acetyl-CoA from pyruvate. Tryptophan synthase Two Acetyl-CoA formed by this enzyme complex is the precursor for the TCA cycle. Details of Pyruvate dehydrogenase Multienzyme complex this complex are discussed in Chapter 3.

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1.6 MULTISUBSTRATE REACTION

Multisubstrate reaction is considered as a sequence of individual steps, each of which obeys the single and double displacement model. Pyruvate dehydrogenase is the best example for this reaction (as explained in Chapter 2).

1.7 ENZYME LOCALIZATION—HISTOENZYMOLOGY

Histoenzymology literally relates biochemical activity with histology.

1.7.1 DEFINITION OF HISTOENZYMOLOGY Histoenzymology is defined as localization of an enzyme and measurement of enzyme activity in defined cells lying within a complex histology. The subcellular location of enzymes may be revealed by microscopy, after fixation and staining.

1.7.2 APPLICATIONS Histoenzymology is used to investigate biochemical cellular changes that may be induced by a drug, a disease, or a toxic agent. Hence, enzyme activity in defined cells is used as biomarkers. Uses of histoenzymology are summarized in Table 1.7. For example, alkaline phosphatase can be identified by the formation of a black precipitate. In this reaction b-glycerophosphate is used as a substrate in the presence of lead ions. Acid phosphatase present in the tissue acts on the substrate catalyzing the formation of glycerate and phosphate. Phosphate immediately reacts with lead ions forming insoluble lead phosphate, which is allowed to react with ammonium sulphide. Ammonium sulphide reaction produces a black precipitate of lead sulphide. These reactions are given as follows: 2– b-glycerophosphate Glycerate + PO4 2– 2+ PO4 + Pb PbPO4 (NH42 ) S PbPO4 PbS black color Black precipitate is used as cell surface marker. Black particles generated by acid phosphatase are used as a marker of lysosomal activity. Similarly, each enzyme produces a specific color after a specific reaction. Black color produced by succinate dehydrogenase is used as a marker of mitochondrial activity. Blue color produced by glyceraldehyde-3-P dehydrogenase is used as a marker of EMP pathway. Color produced by hydroxylacyl dehydrogenase activity is used as a marker of fatty acid utilization. Na+ K+ATPase activity is used as a marker of cell membrane activity. Details of the substrate, coenzyme, buffer, pH, and other components in the reaction mixture used in histoenzymology are summarized in Table 1.8

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Table 1.7 Uses of histoenzymology Enzymes Uses Alkaline phosphatase Cell surface marker Acid phosphatase Cell surface marker Succinate dehydrogenase Marker of mitochondrial activity Glyceraldehyde-3-P dehydrogenase Marker of EMP pathway Hydroxylacyl dehydrogenase Marker of fatty acid utilization Na+ K+ATPase Marker of cell membrane activity

Table 1.8 Reaction mixture used in histoenzymology Enzymes Substrate/coenzyme Buffer-pH Other components

Alkaline phosphatase Na-b-glycerophosphate Barbitone-9.4 Calcium chloride, Magnesium sulphate, Cobalt nitrate, Ammonium polysulphide Acid phosphatase Na-b-glycerophosphate Acetate-5.0 Lead nitrate, Hydrogen sulphide Succinate dehydrogenase Sodium succinate Phosphate-7.8 Phenazine methosulphate and Nitroblue tetrazolium Glyceraldehyde-3-P Fructose –1,6– Glycyl glycine–8.0 Phenazine dehydrogenase bisphosphate/aldolase/ methosulphate & NAD Nitroblue tetrazolium Hydroxylacyl Butyryl cysteamine/ Glycyl glycine–8.0 Sodium nitroprusside, dehydrogenase NAD menadione, neotetrazolium chloride Na+ K+ATPase Disodium adenosine-5¢ Tris–7.4 Sodium acetate, tri-P/lead ammonium Sodium chloride, citrate/acetate Magnesium chloride, Potassium chloride Lactate dehydrogenase Sodium lactate/NAD Glycyl glycine–8.0 Phenazine methosulphate & Nitroblue tetrazolium Glucose-6-P Glucose-6-P-disodium Glycyl glycine–8.0 Phenazine dehydrogenase salt/NADP methosulphate & Nitroblue tetrazolium Guanylate lyase Guanosine-tri-P Tris–7.4 Sodium acetate, lithium salt Sodium fluoride, Magnesium chloride

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1.8 ENZYME ASSAY

Enzyme activity can be assayed by the following methods: ∑ Direct assay ∑ Coupled assay/linked assay (two-step and three-step coupled assay)

1.8.1 DIRECT ASSAY In the direct method, enzyme activity is assayed by directly measuring the absorbance of light by the product.

Dehydrogenases Dehydrogenase can be assayed by measuring the absorbance of NADH at 340 nm. Dehydrogenase Substrate (reduced) + NAD+ Product (oxidized) + NADH + H+

Absorption at 340 nm Amount of light absorbed by NADH at 340 nm is proportional to dehydrogenase activity.

1.8.2 TWO-STEP COUPLED ASSAY OR LINKED ASSAY In some cases neither the product nor the substrate can be quantitatively measured by determining the light absorption. In such cases, the enzyme reaction is coupled to a second reaction, which can very well produce a light-absorbing product. The product of the first reaction becomes the substrate for the second reaction. The amount of light absorbed by the product of the second reaction is proportional to the enzyme activity. This method is called coupled assay or linked assay. This method is generally not applicable to measurements in crude preparations, which contain NADH-oxidases and other NADH-utilizing enzymes. One important example is the pyruvate dehydrogenase complex.

Hexokinase and glucose oxidase Hexokinase can be assayed by coupling to a dehydrogenase reaction as shown in Fig. 1.11. Hexokinase converts glucose into glucose-6-P, which cannot absorb light. However, when glucose-6-P is coupled to a second enzyme-glucose-6-P dehydrogenase, 6-P-gluconolactone and NADPH are formed. Absorption of NADH measured at 340 nm is proportional to hexokinase activity.

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Glucose Hexokinase ATP + Mg2+

ADP + Glucose-6-P No coloration

Glucose-6-P- NADP dehydrogenase 6-P-gluconolactone + NADPH

Absorption at 340 nm

Proportional to hexokinase activity

Fig. 1.11 Coupled assay of hexokinase

Coupled assay of glucose oxidase is illustrated in Fig. 1.12. Glucose oxidase converts glucose to gluconic acid (a non-absorbing product). Gluconic acid is coupled to peroxidase via hydrogen peroxide and a color-producing dye, 4-amino antipyrine. The dye is oxidized to colored form quinoneimine. Absorbance of quinoneimine is proportional to glucose-oxidase activity.

Glucose + O2

Glucose oxidase H2 O

Gluconic acid + H22 O Peroxidase + 4-aminoantipyrine (colorless) Qunoneimine (colored)

Absorption at 500 nm

Proportional to glucose oxidase activity

Fig. 1.12 Coupled assay of glucose oxidase

In the three-step coupled assay method the logic is similar to the two-coupled assay, but it involves three inter-linked sequential reactions. Activity of the enzyme catalyzing the first reaction is assayed using the product of the third reaction.

Cha 1.pmd 21 9/9/2010, 6:54 PM 22 Enzymology Uricase and cholesterol esterase As shown in Fig. 1.13, in the first reaction uricase converts uric acid into allantoin and H2O2. In the second reaction, H2O2 interacts with ethanol in the presence of catalase, forming acetaldehyde and water. In the third reaction, acetaldehyde combines with NADP+ in the presence of dehydrogenase, forming acetate, NADPH + H+. Absorption of NADPH at 340 nm is proportional to uricase activity. As shown in Fig. 1.14, in the first step of the assay cholesterol esterase hydrolyzes cholesterol ester into cholesterol and fatty acid. In the second reaction, cholesterol oxidase oxidizes cholesterol into 4-choestenone, simultaneously liberating H2O2. In the third reaction, peroxidase catalyzes the reaction of H2O2 with phenol and 4-aminoantipyrine, forming quinoneimine. Absorption of quinoneimine at 500 nm is proportional to the activity of the first enzyme, cholesterol-oxidase.

Uricase Step 1: Uric acid + O2 +H2 O Allantoin + CO2 +H22 O

Catalase Step 2: H22 O + ethanol H2 O + Acetaldehyde

+ Dehydrogenase + Step 3: Acetaldehyde + NADP HO2 + NADPH + H

Absorption at 340 nm

Proportional to uricase activity

Fig. 1.13 Three-step coupled assay of uricase

Esterase Step 1: Cholesterol ester + H2 O Cholesterol + Fatty acid

Oxidase Step 2: Cholesterol + O2 4-cholestenone + H22 O

Peroxidase Step 3: 2H22 O + Phenol + 4-aminoantipyrine Quinoneimine + H2 O

Absorption at 500 nm

Proportional toesterase activity Fig. 1.14 Three-step coupled assay of cholesterol esterase

1.8.2 INSTRUMENTAL TECHNIQUES USED IN ENZYME ASSAY Various techniques used in enzyme assay are as follows: ∑ Spectrophotometry ∑ Fluorimetry

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∑ Luminometry ∑ Radiometry ∑ Manometry ∑ Enthalpimetry/Micro-calorimetry

Spectrophotometry Spectrophotometry is a technique in which the amount of a substance is quantified using the light absorbed by the substance (absorbance, A) based on Beer-Lambert’s Law. Thus, if the substrate or the product of an enzyme-catalyzed reaction absorbs light then the amount of the light absorbed is proportional to the enzyme activity. The absorbance, A is given by a simple formula,

A = log10(I/I0)

where I0 is the intensity of the incident light and I is the intensity of the light transmitted through the sample. According to Beer-Lambert’s law A = ecl where e is the molar extinction coefficient, c is the molar concentration and l is the length of the light path through the sample in cm. Spectrophotometry is used in the assay of enzymes using NAD+ or NADH as coenzyme.

Fluorimetry Fluorescence is a process by which molecules absorb light of a particular wavelength and re-emit light of a longer wavelength. Fluorimetry involves measurement of the fluorescence of the product of an enzyme catalyzed reaction using a fluorimeter. Fluo- rescence is related to the enzyme activity. Reduced forms of nicotinamide coenzymes- NADH and NADPH absorb light at 340 nm and fluoresce at 460 nm. Therefore, enzymes utilizing NAD and NADH as coenzymes can be assayed by measuring the fluorescence of the products—NADH and NADPH. Oxidases and dehydrogenases can be assayed by this technique.

Substrate + NAD/NADP Oxidase/Dehydrogenase Product + NADH/ NADPH (reduced) (oxidized)

Fluoresce at 460 nm

Fluorescence proportional to enzyme activity

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For example, b-galactosidase can be assayed using a synthetic substrate 4-methyl umbelliferyl-b-D-galactoside. Derivatives of 4-methyl umbelliferone are also used in the assay of esterases, phosphatases, and sulphatases. Lipase can be assayed by using a non fluorescent substrate dibutyryl fluorescein, which is hydrolyzed to a fluorescent product, fluorescein. Lipase Dibutyryl fluorescein Fluorescein Fluorescence Intensity proportional to lipase activity

Luminometry Some of the products emit light during the course of the reaction. This process is called luminescence. This phenomenon is called bioluminescence for a biological reaction and chemiluminescence for a chemical reaction. The intensity of luminescence is measured by a luminometer, which in turn is proportional to the enzyme activity. For example, luciferase acts on its substrate to produce a luminescent product—oxyluciferin. The intensity of the luminescence is proportional to the luciferase activity.

Luciferin + ATP Luciferyl adenylate + PPi Luciferase Luciferyl adenylate + O2 Oxyluciferin + AMP + LIGHT

Intensity proportional to luciferase activity

Radiometry Radiometry involves assay of enzymes whose substrate is radioactively labeled. In such cases the product will be radiolabeled and the radioactivity is proportional to the enzyme activity. b-emitters like tritium (3H), carbon (14C), phosphorous (32P), sulphur (35S), and iodine (135I) are used as radiolabels and the radioactivity is measured by using Geiger Muller counter, or more preferably scintillation counter. For example, acetyl choline esterase is assayed by using the 14C labeled substrate—acetylcholine, and measuring the radioactivity of the 14C-labeled product—acetate. Acetylcholine esterase 14C–Acetylcholine Choline + 14C–Acetate

Radioactivity

Measured by scintillation counter

Proportional to acetylcholine esterase activity

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Manometry is used to assay enzymes whose substrate or product is a gas. This technique makes use of a pressure-measuring apparatus, called a manometer, for measuring the pressure changes produced during the reaction. This method is used to assay the reactions where gas is consumed or liberated. For example, glucose oxidase, which uses molecular oxygen as a substrate or decarboxylases, which produce carbondioxide as a product, may be assayed by manometry. Sometimes, reactions that produce acid can be assayed by adding bicarbonate to the reaction mixture and measuring the evolu- tion of carbon dioxide.

Enthalpimetry

Enthalpimetry, also called microcalorimetry, is an assay technique that measures the amount of heat liberated during an enzyme-catalyzed reaction, with the help of a micro- calorimeter provided with a highly sensitive temperature-sensor-like thermistor. Usu- ally, the product of the primary reaction may be linked to a secondary reaction with a buffer to liberate more amounts of heat than the primary reaction. This results in higher sensitivity. Hexokinase activity can be measured by coupling the primary reaction (phos- phorylation reaction) to a Tris-buffer reaction via protons (H+). Primary reaction Glucose + ATP Glucose-6-phosphate + ADP + H+ (DH = – 28 kJmol–1) Secondary reaction H+ + Tris TrisH+ (DH= – 47 kJmol–1) Here, the enthalpy of the secondary reaction is higher than the primary reaction, which can be measured with high sensitivity.

1.9 PURIFICATION OF ENZYMES FROM NATURAL RESOURCES

Purification of enzymes from their sources is a crucial part of enzymology, which is needed for investigation on the structure and function of enzymes. This section describes different steps involved in enzyme purification like: choosing the sources used for extracting enzymes, methods used for separating enzymes and other impurities.

1.9.1 NEED FOR ENZYME PURIFICATION Enzyme purification is done in order to isolate an enzyme with maximum catalytic activity or specific activity.

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1.9.2 STEPS IN ENZYME PURIFICATION Enzyme purification is a multistep process as shown below: Step 1 Choosing of a suitable source and extraction of enzyme from the source Step 2 Removal of whole cells (undisturbed cells) and cell debris from the enzyme extract Step 3 Removal of nucleic acids and lipids Step 4 Concentration and primary purification of enzyme Step 5 Final purification by column chromatography and quality check These five steps in enzyme purification are shown as a flow-sheet in Fig. 1.15.

Step 2 Removal of whole cells and cell debris Enzyme Crude – By centrifugation extract enzyme – By filtration – By partition

Step 1 Step 3 Choosing enzyme source and recovery Removal of nucleic acids and lipids of enzyme – By precipitation – By filtration – By nuclease – By centrifugation – By glass wool – By cell disruption

Step 4 Concentration and primary purification Concentrated – By ultrafiltration Dilute enzyme – By precipitation enzyme – By chromatography – By dehydration

Step 5 Final purification – By gel filtration chromatography – By ion exchange chromatography – By affinity chromatography – By hydrophobic interaction chromatography – By chromato focussing – By HPLC

Pure Enzyme

Fig. 1.15 Flow-sheet for enzyme purification

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Step 1 Extraction (initial recovery) of enzyme from natural source A prerequisite for the initial recovery of any enzyme is the identification of a suitable source that is rich in the target enzyme. Enzymes are usually recovered from a microbial source, a plant source, or an animal source.

Microbial source Many enzymes of industrial interest, especially extracellular enzymes, are obtained from microbial sources as they can be cultured in large quantities over a short time period by fermentation. Suitable microbial sources are identified and isolated by screening strategies. Microbial enzymes are often more stable than analogous enzymes obtained from plant or animal tissue. Generally Recognized As Table 1.9 GRAS-certified microbes used for Safe–(GRAS) certified microbes are chosen for en- enzyme sourcing and purification zyme recovery. GRAS-listed microbes are non- Bacteria Bacillus subtilis pathogenic, nontoxic, and generally they do not pro- Bacillus amyloliquifaciens duce antibiotics. GRAS-listed bacteria includes Ba- Lactobacillus species cillus subtilis, B. amyloliquifaciens and Lactoba- cillus species. GRAS-listed fungi for enzyme sourc- Fungi Aspergillus species ing include Aspergillus species, Penicillium species, Penicillium species Mucor, Rhizopium species, and Saccharomyces Mucor species cerevesiae. This is shown in Table 1.9. Enzymes Rhizopium species purified from a range of microbes are shown in Saccharomyces cerevesiae Table 1.10.

Table 1.10 Enzymes recovered and purified from a range of microbial sources Enzyme Microbial source Enzyme Microbial source Lactate dehydrogenase Yeast Asparaginase Escherechia coli Cholesterol esterase Pseudomonas species Glucose-6-P-dehydrogenase Yeast Leuconostac mesentroides Glucose oxidase Aspergillus niger Collagenase Clostridium species Glycerol kinase Candida Mycoderma a-amylase Bacillus subtilis Arthobacter species Asereillus species Phytase Aspergillus flavus Aminoacylase Aspergillus oryzae Aspergillus candidus Bacillus stearothermophilus Hexokinase Yeast Cellulase Trichoderma viridae Lactase Kluveromyces lactis Trichoderma reesei Kluveromyces fragilis Trichoderma koningii Aspergillus niger Penicillium fumiculosum

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Table 1.10 (Contd.) Enzyme Microbial source Enzyme Microbial source Pullulanase Aerobacter aerogenes Protease Bacillus licheniforms Klebsciella aerogenes Bacillus lentus Invertase Saccharomyces species Bacillus alcalophilas Pectinase Aspergillus niger Bacillus Aspergillus fusarium amyloliquifaciens Phytase Aspergillus niger Xylanase Clostridium thermocellum Lipase Humicola languinosa Thermolysin Bacillus thermoproteoliticus Cellulase Humicola insolens Cyclodextrin glycosyl Bacillus megatarium transferase Bacillus circilans Glucoamylase Aspergillus niger Penicillin acylase Kluvera species Glucose isomerase Aerobacter species Aerobacter species Bacillus species Brevibacterium species Lactobacillus brewis Proteus rettgeri Actinoplanes missouriensis Proteus morganii

Microbial enzymes are useful in industrial process as well as for therapeutic purposes. Microbial source of some important enzymes and their applications are given in Table 1.11.

Table 1.11 Applications of enzymes sourced from microbes Enzyme Source Application Streptokinase Hemolytic Streptococci Thrombolytic agent Staphylokinase Staphylococcus aureus Thrombolytic agent Asparaginase Erwinia chrysanthemi Treatment of leukemia Glucose oxidase Aspergillus niger Quantification of blood glucose Alcohol dehydrogenase Aspergillus niger Determination of blood alcohol level Amylase Bacillus species Degradation of starch Aspergillus oryzae Protease Bacilli, Aspergilli Degradation of proteins for food and detergent Cellulase Trichoderma species Degradation of cellulose Aspergillus niger Actinomyces

Cha 1.pmd 28 9/9/2010, 6:54 PM Introduction to Enzymology 29 Heterologous enzymes in microbes Expression of enzymes in cells that are not a natural source of that enzyme is called recombinant enzyme production. Such enzymes are produced by recombinant technology and are called heterologous enzymes. Escherechira coli is the commonly employed organism of heterologous enzyme production. The enzyme expressed accumulates in the cytoplasm from which it can be recovered or the enzyme is secreted into the medium using signal peptide (recombinant DNA technology). Other microbial sources for heterologous production include Lactobacillus lactis, Cornybacterium glutamaticum, Bacil- lus species, and Streptomyces species. Yeast such as Saccharomyces cerevasiae and P. pastoris are suitable candidates for heterologous production of a-1 antitrypsin and streptokinase, respectively. Filamentous fungi are used for producing extracellular enzymes. For example, triglyceride lipase and aspartic protease.

Plant source Plants represent a traditional source of a wide range of enzymes. Plant tissues are chosen as a source for subsequent purification of various enzymes like ascorbate oxidase, phytase, peroxidase, urease, bromealin, amylase, lipase, sucrase, nitrate reductase, aldehyde oxidase, protesse, xanthine dehydrogenase, sulphate oxidase, fructose bisphosphate aldolase, sedoheptulose bisphosphatase, fructose bisphosphatase and papain. The plant source of these enzymes are given in Table 1.12. Among these, papain and ficin (protease) assume much significance because they are the most widely studied enzymes. Like microbes, plants can also serve as a source for heterologous enzymes. For example, a-amylase, chymosin (rennin), cyclodextrin transferase, glucoamylase, lysozyme, xylanase, and phytase are heterologous enzymes expressed in plants. The original source and the plant expression system of some enzymes are shown in Table 1.13.

Table 1.12 Enzymes purified from plant source Enzyme Plant Source Ascorbate oxidase Curcubita species Peroxidase Horseradish Phosphoglycerol pyruvate Maize leaves carboxylase Urease Jack bean Bromealin Pineapple stem/fruit b-amylase Barley Pectin esterase Citrus fruits Phytase Wheat, rye, triticale Papain Latex and leaves and unripe fruits of papaya Ficin Latex of certain tropical trees

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Table 1.13 Heterologous enzymes expressed in plants Enzyme Original source Plant expression system a-amylase Bacillus licheniforms Tobacco Chymosin Calf Tobacco Cyclodextrin Klebsciella pneumoniae Potato Glucoamylase Aspergillus niger Potato Lysozyme Chicken Tobacco Phytase Aspergillus niger Tobacco Xylanase Clostridium thermocellum Tobacco

Animal source Animal tissues are a source of several enzymes of industrial use and therapeutic use. The choice of tissue depends on the extent of expression of a particular enzyme in that tissue. For example, the pancreas is a good source for the purification of digestive enzymes; liver is the best source for recovery and purification of detoxification enzymes (like SOD). Other organs like stomach, placenta, heart, kidney, or cells like erythrocytes can be sources for specific enzymes. Table 1.14 summarizes the list of enzymes purified from animal sources. Methods for initial recovery Any of the above-mentioned sources that are essentially rich in the target enzyme is chosen and the enzymes are recovered by several methods.

Table 1.14 Enzymes purified from animal sources Enzyme Animal Source

Alkaline phosphatase Calf intestine and kidney Acetyl choline esterase Bovine erythrocytes Arginase Beef liver Cholesterol esterase Pig beef pancreas Creatine kinase Rabbit muscle, beef heart, pig heart Glutamate dehydrogenase Beef liver Glycerol-3-P-dehydrogenase Rabbit muscle Uricase Porcine liver Xanthine oxidase Buttermilk Pepsin Mammalian stomach Reinin (chymosin) Mammalian stomach DNAse Mammalian pancreas Chymotrypsin Mammalian pancreas Trypsin Mammalian pancreas Glucocerebrosidase Placenta

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These methods are mentioned in Table 1.15 and discussed below.

Table 1.15 Methods of cell disruption for enzyme recovery Enzyme Method Intracellular enzyme Filtration Cytosolic enzyme Differential centrifugation Subcellular organelle enzyme Differential centrifugation Extracellular enzyme Cell disruption using Secretory enzyme 1. Blender 2. Mortar and pestle 3. Dynomill or bead mill 4. High-pressure homogenizer 5. Osmotic shock 6. Enzymes 7. Detergents or chemicals 8. Sonicator 9. Freeze-thaw cycle

Recovery of extracellular enzymes Most of the microbial enzymes are produced by fermentation and released into the fermentation medium. Such secretory enzymes can be recovered by filtration or by centrifugation of the fermentation medium.

Recovery of intracellular enzymes Intracellular enzymes present within plant cells, animal cells or microbial cells are recovered by cell disruption in which cell architecture is disorganized in the presence of a buffer in order to release the cellular content and form crude extract. Cell disruption can be done by using blenders, homogenizers or other methods, based on the nature of the enzymes. Mechanical methods should be strong enough to break the cell wall and the membrane, but should not physically damage the enzymes. Also, cold conditions are preferable to avoid heat-induced damage during the mechanical operation. Various methods of cell disruption for enzyme recovery are listed in Table 1.15.

Blender A blender has a small chamber equipped with a set of blades of different shapes. The chamber is filled with the specimen and the blender is operated electroni- cally. The blades rotate and exert a shearing force to disturb the cell architecture and release the homogenate. Plant cells and mammalian cells may be homogenized using a blender. Mortar and pestle A small amount of the tissue sample is taken in a mortar and hand- ground with the pestle in the presence of abrasives like fine glass pieces and a buffer to form a homogenate. Plant leaves, liver, kidney, and other organs can be disrupted on a

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small scale by this method. Figure 1.16 shows a picture of mortar and pestle. Dynomill or bead mill Components of a dynomill are shown in Fig. 1.17. It is made up of a chamber provided with a rotating impeller disc. The source tissue and glass beads of 0.5mm diameter are loaded Fig. 1.16 Mortar and Pestle in the chamber. The impeller disc is rotated at high speed at which for small scale cell the velocity is approximately around 10m/sec. The beads collide with disruption the disc and the cells are trapped between the beads and the disc, leading to disruption of cellular architecture.

Sample

Chamber Cell Glass bead Impeller disc Rotating arm

Fig. 1.17 A dynomill High-pressure homogenizer A high-pressure homogenizer is shown in Fig. 1.18. It has an upper chamber and a lower chamber, both connected by a small orifice.

Sample High pressure

Piston Upper chamber (High pressure) Intact cell Orifice

Lower chamber (Low pressure) Disturbed cell (lysate)

Fig. 1.18 High-pressure homogenizer

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The cell suspension is introduced through an inlet into the upper chamber and subjected to a high pressure created by a mechanically driven piston. The cells are thus mechanically pushed through the orifice into the lower chamber, where the pressure drops suddenly. As a result, the cells burst abruptly due to turbulence and shearing force. The crude extract is collected through the outlet. Plant cells and microbial cells can be disturbed by this method. The French Press and the Hughes Press are commer- cially available high-pressure homogenizers that can be used for a large volume of cells. Osmotic shock Animal cells can be lysed by osmotic shock. Cells are placed in a buffer of high osmotic pressure, for example, a buffer with 20% sucrose for half an hour to one hour. Consequently, water molecules diffuse from the cells into the buffer causing the cells to shrink. The shrunken cells are then transferred to a buffer of low osmotic pressure or into distilled water. As a result, water molecules diffuse into the cell causing the cells to swell and rupture.

Table 1.16 Enzymes used in cell disruption Enzymes Specific enzymes interact with a Enzyme Cells disrupted wide range of microbial cells and induce cell dis- Lysozyme Gram-negative bacteria ruption. Lysozyme cleaves the cell wall of Gram- Zymolase or lyticase Yeast cell positive bacteria. Zymolase or lyticase disturbs Chitinase Fungal cell yeast cell. Chitinase is used to disturb the cell wall architecture of fungal cells. This is shown in Table 1.16. Table 1.17 Chemicals used in cell disruption Detergents Sodium lauryl sulphate Detergents and chemicals Detergents and Polysorbate chemicals used to disturb microbial cells for re- Triton covering intracellular enzymes are shown in Table Solvents Toluene 1.17. Detergents like sodium lauryl sulphate, Acetone polysorbate, triton, and solvents like toluene ac- Chaotropic agents Urea etone, etc. and chaotropic agents like urea or guani- Guanidine dine are used to disturb microbial cells on a large scale.

Sonicator Sonicator is a device that generates sound waves. Cells are exposed to high- frequency sound waves of greater than 20 Hz for 30–60 seconds in the presence of ice. Sound waves create a shearing force and disturb the cell architecture. Freeze-thaw cycle The freeze-thaw cycle involves repeated freezing and thawing of cell suspension. This results in intracellular ice crystal growth leading to membrane damage and cell rupture. As a result of cell disruption the enzyme will be present in the form of a crude extract. It should therefore be separated from cell debris and the whole cells.

Cha 1a.pmd 33 9/9/2010, 6:55 PM 34 Enzymology Step 2 Removal of cell debris and whole cells After cell disruption the intracellular enzymes are released into the medium (homogenate) and thereafter the cell debris and whole cells can be removed by centrifugation, filtration, or aqueous two-phase partitioning. Details of the methods used for removing cell debris during enzyme purification are given in Table 1.18.

Table 1.18 Methods used for removing cell debris during enzyme purification Method Type Apparatus/materials involved

Centrifugation Continuous flow centrifugation Continuous-flow centrifuge, Disc-type centrifuge, Hollow-bowl centrifuge, Basket centrifuge

Filtration Fiber depth filtration Cellulose fiber, glass fiber Polymer membrane filtration Cellulose acetate membrane system Modern cartridge filtration Polytetrafluoro ethylene membrane system Nylon membrane system

Partitioning Double aqueous phase Two incompatible water soluble partitioning system polymers

Centrifugation Centrifugation is usually done in a continuous flow centrifuge. The homogenate is continuously pumped and the clarified solution is continually collected. The deposited solids (whole cells and cell debris) can be removed from the centrifuge bowl by periodically stopping centrifuge and manually removing the pelleted material. Modern centrifuge allows intermittent discharge of solids through a discharge valve or a periph- eral nozzle. Filtration Filtration is done through a depth filter, a membrane filter, or a modern cartridge filter. A depth filter is made up of randomly cross-linked cellulose fibers or glass fibers forming a mesh work of channels. It retains whole cells, cell debris, denatured proteins, aggregates, and precipitates not only on the surface but also within the depth of the filter. The enzyme is then filtered out. Membrane-like sheets of polymers like cellulose acetate, nylon, or polytetrafluoro ethylene with pore size 0.02–10 m are used to filter cell debris under pressure. This method is also called microfiltration. Modern cartridge filtration system involves filter sheets of different pore size folded into a single cylindrical cartridge system by joining both ends. This is useful in efficient filtration. This method is shown in Fig. 1.19.

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5mm

1mm

0.45m m

Fig. 1.19 Modern cartridge filtration system Aqueous two phase partitioning Aqueous polymer solutions are similar to solutions of some polar organic solvents like alcohol, acetone, and acetonitrile. These organic solvents are completely miscible in water, and so are the aqueous polymers like polyethyleneglycol and polyvinylpyrrolidone. In both cases (aqueous organic solvent and aqueous polymer solutions) the phase partitioning can be induced by salt addition of salt. Aqueous two-phase partitioning is shown in Fig. 1.20.

Less polar/less dense phase Soluble enzyme

More polar/more dense phase Cell debris

Fig. 1.20 Aqueous two-phase partitioning of cell debris and enzyme

When the homogenate is passed through such a partitioning system, debris settles to the lower phase, while soluble enzymes are retained on the less dense upper phase. Subsequently, the two phases are separated. Step 3 Removal of nucleic acids and lipids Nucleic acids present in the homogenate are removed by precipitation with cationic polymer-like polyethylenimine, and subsequent separation by centrifugation or filtration. Compounds like streptomycin, protamine, magnesium chloride, or manganese chloride can also be used for precipitation. Alternatively, treatment with nucleases is also used. Removal of lipid is aided by passing the homogenate through glass wool. The solution obtained after step 3 is called crude enzyme. Step 4 Concentration of enzymes The crude enzyme obtained in the third step is more dilute and hence it is concentrated by various methods like ultrafiltration, precipitation, and dehydration. The principles of these methods are summarized in Table 1.19.

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Ultrafiltration This method is used to concentrate and purify the enzyme preparation by removing impurities of smaller size. Membranes of cellulose acetate, cellulose nitrate, polyvinyl chloride, and polycarbonate with pore size of 1 to 20 nm are used for ultrafiltration. The pore size should be less than the molecular size of the target enzyme. The crude preparation is passed through the membrane filter and pressure is applied using nitrogen gas and peristaltic pump. Molecules with low molecular size than the filter cut-off pore size pass through the ultra filter, while high molecular size enzymes are concentrated and retained within the filter.

Table 1.19 Methods used for concentration of enzymes Method Principle leading to concentration Apparatus Ultrafiltration Filtration under pressure based on Polymer membrane like molecular weight polyvinyl acetate Precipitation Increased protein-protein interaction by Ammonium sulphate salting in and salting out. Precipitation solution below 0°C by lowering dielectric constant Organic solvents of aqueous solution Dehydration Removal of water from enzyme by Sephadex G-25 beads adding sephadex

Precipitation Proteins in crude solution are precipitated using neutral salt, usually ammonium sulphate. Addition of a specific quantity of ammonium sulphate to the crude solution increases the protein solubility. This is called Salting in. However, when salt concentration in the crude solution is further increased above an optimal level the salt molecules effec- tively compete with protein molecules for water of hydration. This promotes increased protein-protein interaction, eventually leading to precipitation. This process is called Salting out. A Diagrammatic representation of salting out is shown in Fig. 1.21.

Native protein + + + – – – + + – – ++ –– + + – – + + + – – – Ammonium sulphate

Protein precipitate

Fig. 1.21 Precipitation of protein by salting out

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Addition of various organic solvents like ethanol, acetone, isopropanol, or diethyl ether at or below 0°C precipitates protein from the solution by lowering the dielectric constant of the aqueous solution. Thermal denaturation can be carried out to precipitate contaminant proteins and nontarget proteins. This is used if the target proteins are extremozymes. Adding dry sephadex Addition of dry sephadex G-25 beads to crude preparation results in the entry of water into the beads, while the enzymes that are too large to enter into the beads remain concentrated.

Step 5 Final purification by column chromatography Column chromatography makes use of a column packed with a stationary phase into which the sample to be purified and the mobile phases are passed through, and the sample separated. In this technique, the separation is based on various principles like gel filtration or exclusion based on molecular size, ion exchange mechanism based on surface charge, biological affinity based on specific biological interaction, high pressure or hydrophobic interaction. This section explains different types of column chromatography principles used in enzyme purification.

Gel filtration chromatography Gel filtration chromatography is also called gel permeation chromatography, or gel exclusion chromatography, or molecular sieve chromatography. It is used to separate and purify enzymes based on molecular size exclusion through a gel bead column. A column of porous gel beads is used as a stationary phase, while a liquid distributed inside and outside the gel pores (sieves) act as a mobile phase. The principle of gel filtration chromatography is shown in Fig. 1.22.

Sample

Gel bead Low molecular weight molecule Intermediate molecular weight molecule High molecular weight molecule

Fig. 1.22 Principle of gel filtration chromatography (the pore size of the gel bead can be chosen to retain the target enzyme to be purified)

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When a sample mixture is passed through such a gel filtration column, molecules are distributed between the inner and the outer mobile phase depending on their molecular size. Compounds of high molecular size are completely excluded from the sieve and come out at a faster rate, while compounds of low molecular size are completely retained within the inner mobile phase of the sieve and come out at a slower rate after elution. Compounds of intermediate molecular size pass through the interstitial space between the gel beads and come out at a moderate speed. If the pore size of the gel beads is chosen to retain the enzyme molecule, the latter can easily be purified and recovered by elution. Porous glass granules or porous gel beds of polymeric organic compounds are used for enzyme purification by gel filtration chromatography. This is shown in Table 1.20. In Fig. 1.23 Gel filtration addition, this method is also used to determine the molecular weight of chromatography enzymes and other proteins. Some amount of water and low molecular equipment weight impurities can be removed using sephadex G-25 column. A picture of the gel filtration chromatography equipment is shown in Fig. 1.23.

Table 1.20 Gel materials used for enzyme purification by gel filtration chromatography Gel Trade Name Agarose Sepharose Biogel-A Polyacrylamide Biogel-P Sephacryl Dextran Sephadex Sephacryl Polystyrene Biobead

Ion exchange chromatography Enzymes with net positive or negative charge are separated and purified due to the at- traction between the charged group of the target molecule and the oppositely charged group present in the solid matrix stationary phase called the ion exchangers. For example, sulphonated polystyrene, carboxymethyl cellulose, and DEAE cellusose (diethylaminoethyl cellulose). Enzyme purification by ion exchange chromatography is shown in Fig. 1.24.

Ion change resin Buffer I C + Binding pH I + C E E

I – Covalently bound matrix ion – Enzyme to be purified E C – Electrostatically bound counter ion

Fig. 1.24 Enzyme purification by ion exchange chromatography

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Ion exchangers are cross-linked inert matrix containing covalently bound ions, which in turn is electrostatically bound to exchangeable counter ions. Matrix that has covalently bound positive ion, which in turn is electrostatically bound to a negative counter ion is (+) called an anionic exchanger. For example, DEAE cellusose: OC2H5–N H(C2H5)2. Matrix that has covalently bound negative ion, which in turn is electrostatically bound to a positive counter ion is called as cationic exchanger. For example, carboxymethyl (CM) (–) cellulose: –OCH2CO 2 . The DEAE exchanger is highly positively charged at pH 6–8, so DEAE cellulose is most useful for enzymes that are negatively charged in this range. Carboxy/methyl cellulose is highly negatively charged, at around pH 4.5, hence CM cellulose is applicable in purifying enzymes that are positively charged at around pH 4.5. This is shown in Fig. 1.25.

+– – + I – Matrix ion I C I C C – Counter ion

Fig. 1.25 Ion exchangers used for enzyme purification Ion exchangers are treated with 0.5N NaOH and 0.5 N HCl and allowed to swell. The swollen exchanger is suspended in water to expose the functional group and equilibrated with suitable counter ions. Excess counter ions can be removed by water-wash and the exchanger packed into the column. The column is equilibrated with a low concentration buffer of suitable binding pH at a rate of 60 ml/hr in order to allow uniform diffusion of the sample into the column and to get a good resolution. A buffer with a pH that is one unit above or below the isoelectric point of the enzyme to be separated is used as a mobile phase. A crude sample is dissolved in the mobile phase and applied on the column and dif- fused in it. At this stage, the charged group of the sample compound undergoes an exchange with the counter ion and adsorbs reversibly to the column. Bound enzyme is eluted by changes in either salt concentration or pH. NaCl salt can be used for elution. In this procedure, as the concentration of the salt (NaCl) increases, the enzyme protein is displaced from DEAE cellulose by anion (Cl–) and from CM cellulose by cation (Na+). In pH elution, the pH is altered over the relatively narrow working range and the proteins are eluted as their isoelectric point is reached, since they then have no net charge to bind the resin.

Affinity chromatography Affinity chromatography is the best method for enzyme purification. This method involves separation of enzyme molecules based on their specific noncovalent reversible biological interaction towards ligands, which are immobilized in a solid stationary phase.

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Matrix Matrix such as agarose, dextran, polystyrene, and polyacrylamide are used as the stationary phase. The stationary phase cho- Spacer arm sen should have a suitable chemical group to bind with the ligand. Ligand The stationary phase is first activated by treatment with cyano- gen bromide. Ligands possessing chemical groups like amino, car- boxyl, thiol, or hydroxyl are covalently immobilized at the station- Fig. 1.26 Affinity column used for enzyme purification by ary phase by means of spacer arms (hydrophobic or hydrophilic affinity chromatography compounds and packed into a column. A column with a stationary phase, a ligand, and a spacer arm can be collectively called an affinity column. This is shown in Fig. 1.26. Tryptophan is used as a ligand for purifying chymotrypsin. (Tryptophan is the substrate for chymotrypsin and capable of binding, but not hydrolyzing, significantly, the enantiomeric substrate analog.) Avidin can be used for separating and purifying biotin containing enzymes because it has a high affinity towards biotin. Metal ions are used as a ligands for superoxide dismutase, (so-called metal affinity chromatography) because the histidine residue of the enzyme forms coordinate covalent bonding with the metal. Cibacron blue dye is used as a ligand for kinases, dehydrogenases, and DNA polymerase (so-called dye ligand chromatography). Dye ligands may act as substrate analogs, offering affinity interactions with their corresponding enzymes. After binding the enzyme with its ligand the affinity column is percolated with a suitable buffer. As a result, enzymes bound to a ligand are retained within the column, while other nonspecific impurities are washed away. Subsequently, enzyme molecules are eluted by percolating the column with a deforming buffer at a pH, that a changes the characteristics of the enzyme and no longer allows it to bind to the immobilized ligand, or by the use of a competitive counter-ligand like reversible inhibitor, a for which the enzyme has more affinity than the column ligand. An overview of enzyme purification by affinity chromatography is shown in Fig. 1.27.

New covalent binding + sample Affinity column

Buffer with Substrate Second Affinity Gradient pH gradient Affinity elution elution or ligand elution (or) Reversible ionic strength infibitor gradient

+ + +

Fig. 1.27 An overview of enzyme purification by affinity chromatography

Cha 1a.pmd 40 9/9/2010, 6:55 PM Introduction to Enzymology 41 Hydrophobic interaction chromatography Hydrophobic interaction chromatography is a type of adsorption chromatography. It involves separation of compounds based on the difference in interaction between the surface specific hydrophobic amino acids of the protein with a solid support called adsorbents. Hexyl, octyl, or phenyl groups supported on an agarose column can be used as adsorbents. Enzymes initially precipitated using ammonium sulphate (step 4) have their hydrophobic amino acid residues on the surface of the protein exposed. The precipitate is then dissolved in a buffer. The pH of the buffer should be equal to the isoelectric point of the protein. The sample is then loaded to the column at high ionic strength. At high ionic strength, nonpolar amino acids form hydrophobic interactions with the column package. Thus, protein can be separated based on the difference in hydrophobic interaction with the adsorbent. The column is eluted with the buffer by continuously decreasing the ionic strength or increasing the pH, as this will decrease the hydrophobic interaction and detach the protein from the column. Alternatively, the column may be percolated with a nonionic detergent such as Tween 20 or Triton × 100. As the adsorbent has greater affinity for the detergent than for the protein, the latter can be eluted out.

HPLC Conventionally, chromatography is not carried out under high pressure. In a unique technique called HPLC (high pressure liquid chromatography), purification and separation can be done under high pressure using microparticulate stationary phase of diameter, based on any one of the principles described for other chromatography. Here the high pressure is used in order to increase the flow rate and to decrease back pressure and get a better resolution than that of low pressure chromatography (conventional chromatography). After the final step of purification by chromatography, the degree of purification of the enzyme can be calculated from the specific activities of the crude and purified extracts using the following formula: Specific activity of purified extract Degree of purification = Specific activity of crude extract Percentage recovery of any enzyme can be calculated from the total units in the crude and purified extracts by using the following formula. Total units in purified extract % recovery = ×100 Total units in crude extract

1.10 FRACTIONATION AND CHARACTERIZATION OF PROTEINS (ENZYMES)

Proteins can be fractionated by various methods and various properties like molecular weight, electrophoretic mobility, isoelectric point, and charge to mass ratio can be determined. Fractionation and characterization can be done by the following methods:

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1.10.1 ANALYTICAL ULTRACENTRIFUGATION Analytical ultracentrifugation is done to find purity, sedimentation rate, molecular weight, and conformational change. An analytical ultracentrifuge operates at a very high speed of 70,000 rpm. This is shown in Fig. 1.28. The protein sample to be characterized is suspended in a suitable medium (solvent) and taken in the analytical cell, which is counter-balanced by the counterpoise cell. The sample is centrifuged for sedimentation. The molecules are fractionated into density zones. A parallel beam of light illuminates the sedimenting particle and gets refracted in the zone, and the refractive index is plotted against the distance of the analytical cell and monitored as the peak. The area of the peak gives the concentration of the sample. The appearance of a single sharp peak indicates purity of the protein, while appearance of an additional peak, shouldering of main peak, and asymmetric peak, represents impurity. Shift in the peak or movement of the peak gives the sedimentation velocity (v) of the particles at time (t). The sedimentation coefficient (S) is calculated using the equation: S = v/w2 r where v = sedimentation velocity w = angular velocity of the rotor r = radial distance moved by the molecules from the axis of rotation S is substituted in the following equation in order to determine the molecular weight M = [RTS/D(1– u)r] where M = Molecular weight of the molecule R =Molar gas constant T = Absolute temperature in Kelvin S = Sedimentation coefficient D = Diffusion coefficient of the molecule u = Partial specific volume of the molecule r = Density of the medium at 20°C

Mirror Peak Motor Armored chamber Photographic plate

Counterpoise cell Rotor Sample tube Analytical cell

Thermistor

Lens

Light source

Fig. 1.28 Analytical ultracentrifuge

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The native protein molecule is compact and the sedimentation rate is faster. How- ever, when there is any conformational change in the native molecule, it becomes disorganized, leading to a decrease in the sedimentation rate. Therefore, the difference in the sedimentation rate, before and after the conformational change gives us an idea of the extent of conformational changes. For example, denaturation of aspartate transcar- bamoylase can be detected.

1.10.2 GEL FILTRATION CHROMATOGRAPHY Proteins of high, low, and intermediate molecular weights are fractionated using distribution coefficient (KD) value and elution volume. The KD value for high-molecular weight molecules is equal to zero, and hence are completely excluded from the inner mobile phase. For low-molecular weight molecules, the KD value is equal to one, and gain com- plete access to the mobile phase within the gel pore. Intermediate-molecular weight molecules have KD values between zero to one and gain partial access to the inner mobile phase with the distribution coefficient value. Elution volume is the volume of liquid (buffer or mobile phase) required to elute a particular compound with a KD value between 0 and 1. It is given by the equation ¥ Ve = Vo + KD Vi

where Vo is the volume external to the gel bead (called void volume). For two compounds having different molecular weight and hence different KD ¢ ¢¢ values, say KD and KD Ve is given by the equation ¢¢ ¢¢ ¥ Ve = KD – KD Vi Molecular weight of the fractionated protein can be determined based on the linear relation between log molecular weight (Mw) and elution volume (Ve). The equilibrated gel column is loaded with standard protein of known molecular weight, e.g. b-galactosidase (116 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa) and lysozyme (143 kDa). Ve is determined for standard protein markers and for unknown protein. A plot of log molecular weight versus Ve of the standard protein is constructed to get a linear relation, as shown in Fig. 1.29. The plot is extrapolated and the molecular weight (Mw) of the test protein is identified.

log Mw Molecular weight (kDa) of unknown protein

Ve (m1)

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1.10.3 ISOELECTRIC FOCUSING Isoelectric focusing (IEF) is a type of gel electrophoresis in which compounds are separated in a gel with pH gradient, based on differences in isoelectric point. Isoelectric focusing gel has preselected pH range. Immobilized pH gradient strips (IPG strips) can also be used for ctrophoresis. IPG strips are available in different lengths to accommo- date various gel sizes and different pH ranges to allow optimal separation. IPG strips reduce the preparation time and reagent waste. Proteins are electrophoresed on IEF gel or IPG strip. Protein molecules with a pH lower than the isoelectric point acquire a net positive charge and move towards the cathode, while those with pH higher than the isoelectric point acquire net negative charge and move towards the anode. Migration continues until the molecules reach a pH region in the gel, which is equal to their isoelectric point. At the region, where the pH of the gel is equal to the isoelectric point of the molecule, the molecules become stationary and exist as zwitter ions, as shown in Fig. 1.30. Thus, proteins could be fractionated based on their isoelectric point.

IEF gel

––– °°° +++ – PPP PPP PPP P Anionic protein Zwitter + ions P Cationic protein P° Neutral protein High pH Low pH (zwitter ions) pH gradient

Fig. 1.30 Protein fractionation based on isoelectric point using isoelectric focusing

1.10.4 ONE-DIMENSIONAL PAGE One-dimensional PAGE is usually done using two different gels of different pore size; upper stacking gel with larger pore size and lower separating gel with smaller pore size. The secondary structure of protein is stabilized by hydrophobic interaction, hydrogen bond, and disulphide bond. So the secondary structure of protein is disturbed by the addition of denaturing agents like SDS, b-mercaptoethanol, and urea in order to aid electroophotetic mobility. Disturbance of secondary structure leads to increase in intrinsic viscosity and increased entropy. Function of denaturing agents are shown in Table 1.21.

Table 1.21 Role of denaturing agent in protein fractionation Denaturing of agent Function SDS Disturbs hydrophobic interaction Confers negative charge to the protein that is proportional to the molecular size b-mercaptoethanol Break disulphide bridges Urea Break hydrogen bonds

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The denatured sample is loaded to the upper stacking gel where protein molecules migrate down towards the anode and are tightly stacked into a sharp distinct disc, which in turn enters into the separating gel and gets resolved based on the molecular-sieving property of the gel. Low molecular weight proteins pass through the gel pores easily and migrate faster, while high molecular weight proteins are retarded due to frictional resis- tance and migrates, slowly. To determine the molecular weight of proteins a plot of molecular weight of standard protein markers against distance traveled (electrophoretic mobility) in the gel is constructed to get a linear relation as shown in Fig. 1.31. This plot gives the inverse relationship between the molecular weight and the distance traveled (electrophoretic mobility). The molecular weight range of the marker proteins should cover the molecular weight of the unknown proteins.

log Mw Molecular weight of unknown protein

Distance (mm)

Fig. 1.31 Determining the molecular weight of protein by SDS PAGE using standard markers

1.10.5 TWO-DIMENSIONAL GEL E LECTROPHORESIS Two-dimensional gel electrophoresis separates proteins, first based on their isoelectric point in a horizontal direction and in the second dimension based on their molecular mass in a vertical direction. In this technique, the protein molecules are first separated in an IEFgel with pH gradient based on differences in isoelectric point. The gel is then incu- bated in SDS to denature the protein. The separation in the second dimension by molecular mass is performed in slab SDS-PAGE. Under applied voltage the denatured protein subunits are separated on the second gel, based on mass. Thus, in 2-D PAGE whole proteins are fractionated. This method is useful for characterization of isoelectric point and molecular mass of proteins. If the identity of the protein is desired, usually the method of in-gel digestion is applied, where the protein spot of interest is excised, and digested proteolytically. The peptide masses resulting from the digestion can be determined by mass spectrometry using peptide mass fingerprinting. If this information does not allow unequivocal identification of the protein, its peptides can be subject to tandem mass spectrometry for de novo sequencing.

Cha 1a.pmd 45 9/9/2010, 6:55 PM 46 Enzymology 1.10.6 HPLC/MS Characterization of protein mixtures using HPLC/MS is also called shotgun proteomics and mudpit (multidimensional protein identification technology). In this method peptides (obtained after enzymatic digestion) are fractionated by one or two steps of liquid chromatography. The eluant from the chromatography stage can be either directly introduced to the mass spectrometer through electrospray ionization, or analyzed using MALDI.

1.10.7 MASS SPECTROMETRY Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles. It is generally used to find the composition of a physical sample by generating a mass spectrum representing the masses of sample components. The mass spectrum is obtained by a mass spectrometer. Components of a mass spectrometer are schematically shown in Fig. 1.32.

Ionization Acceleration

Electromagnet Vapourized sample Ionizing filament

Vacuum

Detector Amplifier

Chart recorder

Fig. 1.32 Components of mass spectrometer

Mass spectrometers consist of three basic parts: an ion source, a mass analyzer, and a detector system. The stages within the mass spectrometer are: ∑ Production of ions from the sample ∑ Separation of ions with different masses ∑ Detection of the number of ions of each mass produced ∑ Data analysis

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Mass spectrometry is used for the characterization of proteins. Methods for ionization of whole proteins are electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI), and surface-enhanced laser desorption/ionization (SELDI). Two approaches are used for characterizing proteins: ∑ Top-down approach ∑ Bottom-down approach In the top-down approach, intact proteins are ionized by either of the two techniques described above, and then introduced to a mass analyzer. In the bottom-down approach, proteins are enzymatically digested into smaller peptides using proteases such as trypsin or pepsin, either in solution or in gel, after electrophoretic separation. Other proteolytic agents are also used. The collected peptide products are then introduced to the mass analyzer. When the characteristic pattern of peptides is used for the identification of the protein the method is called peptide mass fingerprinting (PMF). If the identification is performed using the sequence data determined in tandem MS analysis, i.e. analysis without the help of data base, it is called de novo sequencing. Whole protein mass analysis is primarily conducted using either time-of-flight (TOF) MS, or Fourier transform ion cyclotron resonance (FT-ICR). These two types of instruments are preferable here because of their wide mass range, and in the case of FT- ICR, its high mass accuracy. Mass analysis of proteolytic peptides is a much more popu- lar method of protein characterization, as cheaper instrument designs can be used for characterization. Additionally, sample preparation is easier once whole proteins have been digested into smaller peptide fragments. The most widely used instrument for peptide mass analysis are the MALDI time-of-flight instruments as they permit the acquisition of PMFs at high pace (1 PMF can be analyzed in approximately 10 sec). Multiplestage quadrupole-time-of-flight and the quadruple ion trap also find use in this application. Given below is a list of various mass analyzers used for protein characterization. ∑ Scanning mass analyzer ∑ MALDI time-of-flight mass analyzer ∑ Trapped ion mass analyzer ∑ Fourier transform ion cyclotron resonance ∑ Quadrupole mass analyzer Proteins of interest to biological researchers are usually part of a very complex mixture of other proteins and molecules that coexist in the biological medium. This presents two significant problems. First, the two ionization techniques used for large molecules only work well when the mixture contains roughly equal amounts of constituents, while in

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biological samples, different proteins tend to be present in widely differing amounts. If such a mixture is ionized using electrospray or MALDI, the more abundant species have a tendency to ‘drown’ or suppress signals from less abundant ones. The second problem is the difficulty in interpretation of mass spectrum. Enzymatic digestion of a protein gives rise to a large number of peptide products in the form of complex mixture whose mass spectrum is very difficult to interpret. To contend with this problem, Two-dimensional gel electrophoresis (2-DGE) and HPLC/MS are used to fractionate proteins, or their peptide products from an enzymatic digestion.

1.10.8 PROTEIN IDENTIFICATION BY PEPTIDE MASS FINGERPRINTING Peptide mass fingerprinting is used to identify proteins. This method uses the masses of proteolytic peptides as input to a search of a database of predicted masses that would arise from digestion of a list of known proteins. If a protein sequence in the reference list gives rise to a significant number of predicted masses that match the experimental values, there is some evidence that this protein was present in the original sample.

RECENT DEVELOPMENTS Intestinal enzymes are used in controlling obesity. rodents, the team focused on MGAT2, an enzyme An international team has pinpointed an enzyme found in the intestines of mice as well as humans. that determines whether what fat people eat is burnt Mice without the MGAT2 enzyme protein were able off as energy or stored in the body, a breakthrough to eat a high-fat diet, while remaining slim and that raises the prospect of a pill to target the en- healthy. The fat they absorbed was burnt off as enzyme, allowing people to eat without the risk of ergy, rather than stored. (Source: Deccan Chronicle, putting on weight. In their study on laboratory March 17th, 2009).

SUMMARY

Enzymes are biocatalytic proteins. They possess a tion, catalyzed enzymes are classified into six ma- recognition site for recognizing the substrate and jor classes—oxidoreductase, transferase, hydrolase, an active site for catalyzing the reaction of the isomerase, lyase, and ligase. Each major class, in substrate. Activity of enzymes can be expressed in turn, is further classified into subclasses and sub- terms of international units, or Katal, or turnover subclasses. number, or specific activity. Enzyme-substrate interaction takes place either Enzymes possess remarkable properties like by lock and key model (predetermined active site) catalytic power, specificity, regulatability, milder or by induced fit model (substrate-induced reaction condition, reversibility, denaturability, complementarity). Binding of substrate and release and colloidal nature. Enzyme activity is influenced of product during enzyme-catalyzed reaction is by various factors like substrate concentration, tem- explained by the Cleland notation. perature, pH, and radiation. Thus, each enzyme has Histoenzymology refers to the localization of en- its own optimum parameters (like temperature, pH, zymes and the measurement of enzyme activity in optima, and km value). Based on the type of reac- defined cells lying within a complex histology.

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Histoenzymology is used to investigate biochemi- tion of cell debris and whole cells, and concentra- cal cellular changes that may be induced by a drug, tion and final purification by column chromatog- a disease, or a toxic agent. raphy. Enzyme activity in any source can be assayed by Enzymes can be fractionated and characterized three methods: direct assay, coupled assay/linked by several standard methods like SDS PAGE, 2D- assay, and multistep coupled assay. After the as- PAGE, isoelectric focusing, analytical ultracentri- say, the enzyme can be purified from the relevant fugation, gel filtration chromatography, and mass source by initial recovery of the enzyme, separa- spectrometry.

KEY TERMS

Substrate A substance upon which the enzyme Mechanochemical enzymes Enzymes that con- acts. vert chemical energy into mechanical energy are Simple enzyme An enzyme made up of protein called mechanochemical enzymes. For example, alone. ATPase couples hydrolysis of ATP to induce a conformational change in muscle protein. Conjugated enzyme enzyme conjugated to a non- protein part is called conjugated enzyme or ho- Modulators Modulators are small molecules that loenzyme. modulate the enzyme activity. Apoenzyme The protein part of a conjugated en- Activators Activators are modulators that enzyme is called apoenzyme. hance enzyme activity. Cofactors Inorganic prosthetic groups of enzymes Inhibitors Inhibitors are modulators that decrease are called cofactors. the enzyme activity. Coenzymes Organic prosthetic groups are called Active site An active site is the area of the en- coenzymes. zyme to which the substrate binds for the subse- Endoenzymes Endoenzymes are those whose site quent reaction. of synthesis and site of action are similar. For ex- Homotropic enzyme A homotropic enzyme is one ample, metabolic enzymes. that has a structural similarity with the substrate Exoenzymes Exoenzymes are those whose site of and the modulator. synthesis and site of action are different. For ex- Heterotropic enzyme A heterotropic enzyme is ample, digestive enzymes. one that has no structural similarity with its sub- Adaptive enzymes An adaptive enzyme is one strate and its modulator. that is not constantly present in the cell, and in- Regulatory enzymes Enzymes that respond to stead, is produced only when its substrate is present. various cellular metabolic signals and adjust their Constitutive enzymes Enzymes that are always catalytic activity are called regulatory enzymes. present in relatively constant amounts in the cell, Plasma-specific enzyme Enzymes that are syn- regardless of the presence of the substrate, are called thesized in the tissues but present in the blood in constitutive enzymes. equivalent or higher concentration than in the Chromogenic enzymes Enzymes that convert a tissues are called plasma-specific enzymes. For colorless substrate into a colored product are called example, Lipoprotein lipase chromogenic enzymes. Non-plasma-specific enzyme Enzymes that are Light-emitting enzymes Enzymes that convert present in a lower amount in blood than in tissues normal substrate into a glowing light-emitting are called nonplasma-specific enzymes. For example, product are called light emitting enzymes. Pancreatic lipase.

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Homogenous catalysis In homogenous catalysis, Homogenization Disturbance of cell architecture the catalyst and the substrate are in the same phase to release the cell content/cell lysate is called ho- without any phase boundary. mogenization. Heterogenous catalysis In heterogenous cataly- Analytical ultracentrifugation It is the technique sis, the catalyst is in a phase with the surface bound- by which biomolecules are characterized by sedi- ary. For example, hydrogenation of a ethylene in mentation under a very high centrifugal field in a the presence of a metal catalyst like palladium. refrigerated condition. International Unit (IU) One IU of an enzyme is Sedimentation rate The velocity with which the the amount of enzyme that catalyzes the forma- particles sediment during centrifugation is called tion of one micromole of product, in one minute, as sedimentation rate under optimal conditions of pH, temperature, and ionic strength. Sedimentation coefficient It is the sedimentation rate per unit centrifugal field. Katal One Katal is the amount of enzyme catalyzing the conversion of one mole of substrate to Elution volume It is the volume of the liquid product in one second. One Katal = 6 ¥ 107 IU. required to elute a compound with kD value between 0 to 1. Turnover number (Kcat) It is defined as the number of substrate molecules converted into product Isoelectric focusing It is an electrophoretic tech- per enzyme molecule, per unit time, when the en- nique in which molecules are separated, based on zyme is fully saturated with the substrate. their isoelectric point. Reactive groups These are the functional groups 2D PAGE It is a technique in which molecules are of the enzyme and the substrate that collides for subjected to two different types of electrophore- the reaction to occur. sis-isoelectric focusing followed by SDS-PAGE. Heterologous enzymes Enzymes that are ex- pKa It is the negative logarithm of the acid disso- pressed by recombinant technology in cells that ciation constant, Ka. Just like the pH, the pKa tells are not natural sources of that enzyme are called the acidic or basic nature of solutions. (pKa < 2 heterologus enzymes. means strong acid; pKa > 2 but < 7 means weak Isoelectric point It is the pH at which the pro- acid; pKa > 7 but < 10 means weak base; and pKa > tein bears no net charge. 10 means strong base.

SOLVED PROBLEMS

1. An enzyme of molecular weight 46000, at a 0.25 moles/min concentration of 1 mg catalyzed a reaction at a velocity = 10–3 mg of 0.25 moles/min under optimum conditions. 0.25 moles/min× 103 Calculate the following: = (a) Specific activity of the enzyme in terms mg of units/ mg protein. 250 units (b) Specific activity of the enzyme in terms = mg protein of units/ mole. (c) Turnover number Calculation of specific activity (in terms of units/mole): Solution 250 units Mw Calculation of specific activity (in terms of unit/mg Specific activity = × mg protein mole protein): V units Mw Specific activity = max = 2.5 ¥ 105 ¥ ¥ mg enzyme g protein mole

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= (2.5 ¥ 105 units/g protein)(4.6 ¥ 104g/mole) reaction is directly proportional to the enzyme concentration in a reaction mixture. = 2.5 ¥ 4.6 ¥ 105 ¥ 104 units/g protein ¥ g/mole i.e. [Enzyme] = Velocity ¥ 9 ¥ = 11.5 10 units/g protein g/mole Therefore, 20 ml of preparation contains = 1.15 ¥ 1010 units/mole enzyme 126 units ×20 l Calculation of turnover number: ml 126units 10–6 g 10–6 g = ×0.02ml m ml 1 g enzyme = = 4 Mw mol/g 4.6 ×10 mol/g = 2.52 units enzyme 1 = ××10–6–4 10 or 4.6 V = 2.52 m moles ¥ min–1 ¥ ml–1 = 0.21 ¥ 10–10 Similarly, 5 ml of preparation contains = 2.17 ¥ 10–11 moles enzyme 126units Substitute this value for [E]t in the turnover number ×5 l formula. ml 126units Vmax = × 0.005ml Turnover number = ml []E t = 0.63 units enzyme 0.25 ×10–6 moles/min = V = 0.63 m moles ¥ min–1 ¥ ml–1 2.17 ×10–11 moles enzyme 3. 10 l of a crude enzyme extract from a tissue 0.25 10–6 ×10 –11 moles/min = × catalyzed a reaction at a rate of 0.10 moles /min under 2.17 moles enzyme standard optimum assay condition. 50 ml of the crude extract is fractionated by ammonium sulphate 0.115 ×10–6 ×10 11 moles/min = precipitation and the precipitate when redissolved in moles enzyme 10 ml of the buffer forms a purified extract. 10 l of the = 0.115 ¥ 105 min–1 purified extract catalyzed the same reaction at a rate of 0.18 moles/min. Calculate the percentage recovery = 1.15 × 104 min–1 of the enzyme. 2. An enzyme preparation contains 6 mg of protein/ Solution ml. The specific activity of the enzyme = 21 units/mg protein. Calculate the initial velocity of the reaction in Total units in purified extract % recovery = ¥ 100 a standard 1 ml reaction mixture containing (i) 20 ml of Total units in crude extract preparation and (ii) 5 ml of preparation. Crude enzyme extract contains: Solution 0.10 mole/min Enzyme concentration in a preparation is given as: 10 l [Enzyme] = units/mg protein ¥ mg protein/ml 0.10 mole/min –1–1 21units 6 mg protein = = 10 moles ml min = × 0.01 ml mg protein ml = 10 units/ml [Enzyme] =126 units/ml Total units = units in 50 m l of the extract Consider that the velocity of the enzyme-catalyzed

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Therefore, 1 ml of crude extract contains 30 mg protein. Specific activity of crude extract 10units × 50ml = 500 Total units ml units/ml 15units/ml = = Total units in crude enzyme extract = 500 Total units Mg protein/ml 30mg protein/ml Purified enzyme extract contains = 0.5 units/mg protein 0.18 moles/min Purified enzyme contains 10 l 0.60 moles/min μ 0.18 moles / min –1–1 10 1 = =μ18 moles ml min 0.01 ml 0.60 moles/min = = 60 m moles ml–1 min–1 = 18 units/ml 0.01 m1 Total units = units in 10 ml of the extract = 60 units/ml Therefore, 1 ml of purified extract contains 40 mg protein. Specific activity of purified extract = 18units × 10ml = 180 Total units Units/ml 60 units/ml ml = Mg protein/ml 40 mg protein/ml Total units in purified enzyme extract = 180 Total units = 1.5 units /mg protein Total units in purified extract Degree of purtification = % recovery = ¥ 100 Total units in crude extract Specificactivity of purified extract 15 = =3 180 Specific activity of crude extract 0.5 = ×=100 36% 500 Degree of purification = 3 fold Enzyme recovery = 36% 5. Effect of temperature on Vmax of an enzyme 4. A crude cell-free liver extract contains 30 mg protein/ catalyzed reaction is given below: m ml. 10 l of the crude extract catalyzed a reaction at a Temperature (T °C) V (m moles min–1) rate of 0.15 mole/min under standard optimum assay max condition. 50 ml of the crude extract is fractionated by 25 5.50 ammonium sulphate precipitation. The fraction 30 10.40 precipitating between 20% and 40% saturation was 35 14.15 redissoved in 10 ml buffer. This solution, which forms 40 19.15 the purified extract contains 40 mg protein/ml. 10 l of 45 26.00 the purified extract catalyzed the reaction at a rate of Calculate the Q value. 0.60 mole/min. Calculate the degree of purification 10 obtained by the fraction. Solution

Solution Q10 values are calculated for different 10°C Crude enzyme extract contains: VTat Q = max 2 0.15 moles/min 10 VTmaxat 1 10 l where the difference between T2 and T1 is 10, 0.15 moles/min –1–1i.e T – T = 10°C. = =15 moles ml min 2 1 0.01 ml Therefore, if 35°C and 45°C are considered, then = 15 units/ml

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V at 45°C Therefore, Q = max 10 V at 35°C max 20units= 25units 26.00 0.8 ml ml Q = = 1.83 10 14.15 25× 10–3 units Q between 35°C and 45°C is 1.83. = 25××× 103–1–1 moles litre min 10 litre 6. One gram of fresh weigh of liver contains 20 units of an enzyme with turnover number of Intracellular concentration of enzyme is 4 –1 5 ¥ 10 min . Estimate the intracellular concen-tration Vmax of the enzyme. Turnover number

− Solution 25××× 103– moles litre1– min 1 = Generally it is assumed that fresh tissue has 80% water, 510min× 4–1 all of which is intracellular. That is, fresh tissue has = 5 ¥ 10–3 ¥ 10–4 moles ¥ litre–1 80 ml water = 0.80 ml/g = 5 ¥ 10–7 M 100 g wet wt. Intracellular concentration of enzyme 1 g of tissue has 20 units of enzyme and 1 g of tissue = 5 × 10–7 M corresponds to 0.8 ml of water.

REVIEW QUESTIONS

1. Give evidences to show that enzymes are 8. How is the rate of an enzyme-catalyzed reaction proteins. influenced by substrate concentration, tempera- 2. How does an active site differ from a regulatory ture, and pH? site? 9. Explain the lock and key model and the induced 3. Explain various terms used to measure enzyme fit model for enzyme substrate interaction. activity. 10. What do you mean by histoenzymology? Give its 4. Define holoenzymes, apoenzymes, plasma- spe- applications. cific, non-plasma specific-enzymes, homotropic 11. How is an enzyme activity assayed? enzymes, and heterotropic enzymes. 12. Describe the steps involved in enzyme purifica- 5. What is meant by enzyme specificity? Explain its tion, with a flow sheet. types with suitable examples. 13. Explain the methods involved in fractionation and 6. How are enzymes classified based on the type of characterization of proteins. reaction they catalyze 14. Velocity of a reaction catalyzed by 1 g of enzyme 7. Give the systemic name and the E-C number of of molecular weight 30,666 is equal to 0.166 mole/ the enzyme catalyzing the following reactions: min. UDP-galactose ´ UDP-glucose (i) Find the specific activity of the enzyme in terms of: (a) units/mg and (b) units/mole. Pyruvate + ATP + CO2+ H2O Oxaloacetate + ADP + Pi (ii) Calculate the turnover number. D-glyceraldehyde-3-P ´ dihydroxyacetone [Ans: (i) : (a) 166 units/mg protein; (b) 0.507 × 1010 units/mole enzyme phosphate (ii) 0.5 × 104 min–1]

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15. An enzyme preparation has a specific activity of buffer. This solution, which forms the purified 6.3 units/mg protein and contains 18 mg proteins/ extract, contains 45 mg protein/ml. 10 ml of the ml. Calculate the initial velocity of the reaction in purified extract catalyzed the reaction at a rate of a standard 1ml reaction mixture containing (i) 20 0.75 m mole/min. Calculate the degree of purifica- l and (ii) 5 l of the preparation. tion obtained by the fraction. [Ans: 1.72 fold] –1 –1 [Ans: (i) 22.68 moles × min × ml 18. The following table gives the values of Vmax of an (ii) 5.67 moles × min–1 × ml–1] enzyme-catalyzed reaction at different tempera- 16. Velocity of a reaction catalyzed by 10 l of a crude tures. enzyme extract from a tissue is 0.21 moles /min Temperature (T °C) V (m moles min–1) under standard optimum assay condition. 40 ml max of the crude extract is fractionated by ammonium 25 6.50 sulphate precipitation and the precipitate when 30 12.00 redissolved in 10 ml of the buffer, forms a purified 35 16.50 extract. 10 l of the purified extract catalyzed the 40 22.30 same reaction at a rate of 0.32 moles/min. Calcu- late the percentage recovery of the enzyme. 45 30.00

[Ans: 76.1% recovery] Calculate the Q10 values between (i) 25 °C and 17. A crude enzyme extract from a tissue contains 25 35 °C (ii) 30 °C and 40 °C mg protein/ml. 10 l of the crude extract catalyzed [Ans: (i) 2.46 (ii) 1.85] a reaction at a rate of 0.24 mole/min under stan- 19. A tissue sample has 60 units of an enzyme with dard optimum assay condition. 50 ml of the crude turnover number of 5 × 104 min–1. Calculate the extract is fractionated by ammonium sulphate pre- intracellular concentration of the enzyme. cipitation. The fraction precipitating between 20% [Ans: 9.37 ¥ 10–7 M] and 40% saturation was redissolved in 10 ml

REFERENCES

Bhagvan, N.V. (2002). Medical Biochemistry, 4th ed., Protein Folding, W. H. Freeman and Company, New Harcourt/Academic Press, Massachusetts. York. Binstock, J.F., A. Pramanik, and H. Schulz (1977). “Iso- Garret, R.H. and C.M. Grisham (1995). Biochemistry, lation of a multi-enzyme complex of fatty acid oxi- Saunder’s College Publishing, New York. dation from Escherichia coli.” Proceedings of the Hames, B.D. and N.M. Hooper (2003). Instant Notes nd National Academy of Sciences of the United States Biochemistry, 2 ed., Viva Books Pvt. Ltd., New of America (PNAS), 74(2):492–495. Delhi. Buchholz, K.B. and P.B. Poulsen (1994). In: L. Borross Hyde, C.C. and E.W. Miles (1990). “The tryptophan and J. Tramper (eds), Applied Biocatalysis, synthase multienzyme complex: exploring structure- function relationships with X-ray crystallography Routledge, USA. and mutagenesis” Nature Biotechnology, 1990 Conn, E.E., P.K. Stumpf, G. Bruening, and R.H. Doi Reviews and Perspectives Archive, 27–32. th (2002). Outlines of Biochemistry, 5 ed., John Wiley Lodish, H., A. Berk, S.L. Zipursky, P. Matsudaira, D. & Sons, Singapore. Baltimore, and J. Darnell (2000). Molecular Cell De Robertis, E.D.P. and D.M.F. De Robertis Jr. (2003). Biology, 4th ed., W.H. Freeman and Company, New Cell and Molecular Biology, 8th ed., Lippincott York. Williams and Wilkins, Philadelphia. Murray, R.K., D.K. Garner, P. A. Mayes, and V.W. th Fersht, A. (1999). Structure and Mechanism in Pro- Rodwll (2000). Harper’s Biochemistry, 25 ed., tein Science: A Guide to Enzyme Catalysis and McGraw-Hill, New York.

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Marchiar, P. V. Ballerini, T. Spadling, G. Cera, M.A. Catalytic Proteins, 3rd ed., Oxford University Press, Mussi, J. Moran-Barrio, A. J. Vila, A. M. Vialel, and UK. A. S. Limansky (2008). “A convenient microbio- Roy, R.N. (1998). Practical Physiology, Biochemistry logical assay employing cell-free extracts for the and Biophysics, Viva Books and Allied (P) Ltd., rapid characterization of Gram-negative Calcutta. carbapenemase producers.” Journal of Antimicro- Vijayalakshmi, M.A. (2002). Biochromatography bial Chemotherapy, 62(2):336–344. Theory and Practice, Routledge, USA. Price, N.C. and L. Stevens (1999). Fundamentals of Voet, D. and J.G. Voet (2004). Biochemistry, 3rd ed., John Enzymology: The Cell and Molecular biology of Wiley & Sons, New York.

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