Enzymes Everywhere

ENZYMES

EVERYWHERE

J.G. SHEWALE

National Institute of Science Communication Dr K S Krishnan Marg New Delhi 110 012

CSIR GOLDEN JUBILEE SERIES

ENZYMES EVERYWHERE

Enzymes Everywhere J.G. Shewale

© 1996 National InstitUte of Science Communication First Edition: November 1996 ISBN: 81-7236-141-6 (Paperback)

CSIR Golden Jubilee Series Publication No. 19 Series Editor

Dr Bal Phondke

Volume Editor

Dr Sukanya Datta

Cover Design Illustrations

Pradlp Banerjee Neeru Sharma, Sushila Vohra, Neeru Vijan, Malkhan Sin?;h, Yogesh Kumar, Mohan Singh

Production

Shiv Kumar Marhkan, S P Singh, Ganesh Sahani, Khem Chand, RK. Kaushik, C P S Khari

Printing

Sudhir Chandra Mamgain, GC Porel, Bharat Sing] Rajbir Singh, Pramod Kumar Sharma

FOR SALE IN INDIA ONLY Price: Rs 30.00 (Paperback)

Designed, Printed and Published by National Institute of Science Communication Dr K S Krishnan Marg, New Delhi 110 012

Foreword The Council of Scientific & Industrial Research (CSIR), established in 1942, is committed to the advancement of scientific knowledge, and economic and industrial development of the country. Over the years CSIR has created a base for scientific capability and excellence spanning a wide spectrum of areas enabling it to carry out research and development as well as provide national standards, testing and certification facilities. It has also been training researchers, popularizing science and helping in the inculcation of scientific temper in the country. The CSIR today is a well knit and action oriented network of 41 laboratories spread throughout the country with activities ranging from molecular biology to mining, medicinal plants to mechanical engineering, mathematical modelling to l!letrology, chemitals to coal and so on. While discharging its mandate, CSIR has not lost sight of the necessity to remain at the cutting edge of science in order to be in a position to acquire and generate expertise in frontier areas of technology. CSIR's contributions to high-tech and emerging areas of science and technology are ~ecognised among others for precocious flowering of tissue cultured bamboo, DNA finger-printing, development of non-noble metal zeolite catalysts, mining of polymetallic nodules from the Indian Ocean bed, building an all-composite light research aircraft, high temperature superconductivity, to mention only a few. Being acutely aware that the pace of scientific and technological development cannot be maintained without a steady influx of bright young scientists, CSIR has undertaken a vigorous programme of human resource development which includes, inter alia, collaborative efforts with the University Grants Commission aimed at nurturing the budding careers of fresh science and technology graduates. However, all these would not yield the desired results in the absence of an atmosphere appreciative of advances in science

and technology. If the people at large remain in awe of science and consider it as something which is far removed from their realms, scientific culture cannot take root. CSIR has been alive to this problem and has been active in taking science to the people, particularly through the print medium. It has an active programme aimed at popularization of science, its concepts, achievements and utility, by bringing it to the doorsteps of the masses through both print and electronic media. This is expected to serve a dual purpose. First, it would create awareness and interest among the intelligent layman and, secondly, it would help youngsters at the point of choosing an academic career in getting a broad-based knowlepge about science in general and its frontier areas in particular. Such familiarity would not only kindle in them deep and abiding interest in matters scientific but would also be instrumental in helping them to choose the scientific or technological education- that is best suited to them according to their own interests and aptitudes. There would be no groping in the dark for them. However, this is one field where enough is never enough. This was the driving consideration when it was decide9 to bring out in this 50th anniversary year of CSIR a series of profusely illustrated and specially written popular monographs on a judicious mix of scientific and technological subjects varying from the outer space to the inner space. Some of the important subjects covered are astronomy, meteorology, oceanography, new materials, immunology and biotechnology. It is hoped that this series of monographs would be able to whet the varied appetites of a wide cross-section of the target readership and spur them on to gathering further knowledge on the subjects of their choice and liking. An exciting sojourn through the wonderland of science, we hope, awaits the reader. We can only wish him Bon voyage and say, happy hunting.

PREFACE Almost all people come to know about enzymes during their school days while learning about the process of digestion. However, few 'non-scientific' professionals remain aware of their impc:tance once formal education is over. Enzymes, are one of the few groups of macromolecules that control the existence of living matter on earth. The importance of enzymes becomes evident when one realizes that the synthesis of other crucial regulatory molecules such as DNA, hormones, and neuropeptides are catalysed by enzymes. Though the occurrence of enzymes has been known for centuries, the field of enzymes has seen a revolution only in the last 50 years or so with technological developments in diversified areas like fermentation, separation techniques, genetic engineering, immobilization and bioreactors. All these are covered by a single scientific discipline called Biotechnology. The methodologies are now so advanced that it is possible to selectively alter properties of an enzyme to suit a particular application. My purpose of writing this book is to share th~ new developments in the field of enzymes and their uses with those who do not consciously think about the manifold ,ways in which enzymes touch everyday lives. I hope that the readers come to the conclusion that enzymes play an indespensible role in keeping us healtheir, happier and more content.

ACKNOWLEDGEMENT My desire to write a popt:::larbook crystallised due to the inspiration given by Dr. C. SivaRaman, FNA who has always been a source of motivation for me. I am indeed, grateful to him. After reading the previous books in this series, I had madeup my mind not to miss the opportunity to be associated with a highly learned editor like Dr. G.P Phondke. I sincerely appreciate his suggestions, efforts and concern in bringing out this book in a presentable form. Mr. S.R. Tophkhane, a senior faculty at Institute of Management Education, Pune and a voracious reader has read this book thoroughly. His comments have given a different direction to my writing which is a must for a popular science book such as this. I thank him for his interest and time. Mr. A.K. Basu, Managing Director, and Dr. S.R. Naik, General Manager, R&D, Hindustim Antibiotics Ltd. have nurtured my interests throughout. I express my sincere thanks for their encouragement. I thank Miss. Aruna Deshpande and the most able artists at NISCOM who have created the pictures to make the subject more digestable and to keep-up the spirits of the readers. I also thank all my colleagues at HAL and at NISCOM wno have contributed to this work.

DEDICATED TO My wife MANISHA and

son SHANTANU

Contents

L

Prelude

I

Unknown toBread Known Made Back for to Supermarket the Each Future Other shelf Milking Glossary the Enzymes Our On the Daily Enzyme Curing Looking the Economics Good Ills Spirited Enzymes

86 79 1 30 40 45 51 61 13 24 70 5

what is 'life'? Have you Have everto think wondered ever you paused how

Prelude

the microscopic cells, the units of all life forms carryon their duties and so sustain 'life'? Just a moment's reflection would makeapparent the structural and biochemical complexity inherent in biological systems. This complexity is, however, masked by the synchronized, well orchestrated manner in which the multifarious cellular functions take place. But there is one underlying fundamental need that is common to all cellular functions. Each and every living cell needs energy to survive and conduct its metabolic functions. For providing this energy many chemical reactions take place simultaneously and the sum total of these cellular activities is called metabolism. This is a simple term for a host of complex reactions. For example, we eat food which is broken-down into easily assimilative compounds which are then converted into energy that allows us to carry out our day to day activities. The long drawn process beginning from the moment food is ingested to its final conversion to energy is summed up by the term metabolism. Metabolic

2

ENZYMES EVERYWHERE

Sul1strate

Mr. Enzyme

changes seem very simple but include several complex chemical reactions that are continuously and simultaneously carried out in each and every cell of the body. Trying to mimic similar chemical reactions in the test tubes has not been easy. The obvious question that arises is how are these chemical reactions performed at body temperature? Nature has answered this question in one simple word "Enzymes".

PRELUDE

3

Nearly all chemical reactions in biological system are catalysed by enzymes. A catalyst is a substance that alters the rate of reaction but remains chemically unaltered at the end of the reaction. Most catalysts accelerate the rate of reactions but some, known as negative catalysts, retard the rate of chemical reactions. Catalysts are classified into two groups: chemical catalysts and biological catalysts. Enzymes are biological catalysts or biocatalysts. The prefix 'bia' indicates the origin and the term 'catalyst' indicates the function of the substance. The catalytic efficiency of enzymes is very high. Enzymes can accelerate the rate of a chemical reaction at least 10,00,000 timesr One of the simplest reactions in biological systems involves the addition of a molecule of water to one molecule of carbon dioxide. It is catalysed by an enzyme called carbonic anhydrase. Just one molecule of carbonic anhydrase can add the necessary,water to 105 molecules of carbon dioxide per second and this rate is 107 times faster than one in which no catalyst is involved. The enormous catalytic power of enzymes is attributed to their capacity to bind to substrate molecules in precise configuration and their ability to make and break chemical bonds. Enzymes are unique catalysts and they offer many advantages over chemical catalysts. Enzymes are capable of catalysing reactions at prevailing cellular conditions of acidity or alkalinity, temperature and pressure. They are very efficient and their turnover number is very high which means more of the required end products is formed when an enzyme catalyses a reaction. Enzymes are specific towards substrate. Also, in enzyme catalysed reactions wastage is minimised as side product formation is either nil or minimal. Sometimes, the enzyme may be kept localized on a 'bed' over which the substrate is passed. In such immobilized enzyme preparations the enzyme can easily be separated and reused after the reaction is complete. Thus for large scale or industrial purposes immobilized enzymes spell economic benefits as well. Final products are purer in enzyme catalysed reactions. Such

4

ENZYMES EVERYWHERE

Inside a cell or out of it, enzymes keep on working for man

reactions provide simple, nonhazardous handling conditions and in industrial applications, the pollution load on effluents is reduced. So all in all, industrial houses have a lot to thank enzymes for. *'

agree, is nothing but a fa-

History as everyone will ble agreed upon. It relies

Unknown to

Known

a lot on arguments because the further back in time that one goes, the weaker does the evidence grow. However, the advent of the documentation era has made it easier to substantiate facts. Chronicling the history of enzymes is not an easy job because the constraints that mark the study of history are present here as welL Records of the early days when man first used enzymes or enzyme-like substances are lost in the mists of time. References to the use of substances similar to enzymes for making of cheese are found in Greek epic poems dating from about 800 B.C.The history of man's use of enzymes to suit his own purpose thus stretches quite a long way into the past. However, it was not till 1783, that investigative attempts were made to demonstrate that certain metabolic reactions could be carried out outside the body, in test tubes to be precise, by using catalysts present in cells. Lazzaro Spallanzani, the famous scientist of the time demonstrated that gastric juiCes could digest meat even outside the body. It was much later (1836) that Theodor

6

ENZYMES EVERYWHERE

Theodor Schwarm also formulated the cell theory, which explained that cells are the structural and functional building blocks of living beings

Schwarm named the active component in gastric juice as pepsin. These studies were followed by scientific arguments on whether alcohol fermentation was a monopoly of living cells. Th~ answer came in 1897, when glucose was successfully converted ,to alcohol and carbon dioxide by cell free extract of yeast. It is interesting to note that yeasts were one of the first organisms that were studied in the field of enzymology. The term 'Enzyme' itself was coined in 1876, by Prof.

UNKNOWN TO KNOWN

7

Wilhelm Friedrich Kuhne from Greek words meaning 'In yeast'. Today, the field of enzymology has expanded beyond the expectations of the early investigators. More than 2000 different enzymes are now known and the list is still growing. This has given rise to a piquant situation. Giving a unique name to each and every enzyme so as to ensure for it a distinct identity is proved to be a challenging task. To bring about a degree of uniformity and to lay down internationally respected rules of nomenclature it became necessary to appoint a Commission called "Enzyme Commission". The Commission was appointed in 1956 and its mandate was to provide a systematic platform for the nomenclature of enzymes. It seemed reasonable to classify the enzymes in accordance with the type of reaction they catalyse. Today, all known enzymes are classified into six classes and each class is further divided into subclasses. Each subclass has sub-sub classes listed under it. Any given sub-sub class may have many enzymes each with its own unique number known as tl~e Enzyme Commission (E.C.) number. Trypsin, for example is designated as E.C. 3.4.21.4. The numbering is analogous to the way a student may be identified in school - Class V, Section A, Roll No 46. The difference is that a comma separates the elements here and a point separates the elements in the E.c. number. Usually, however, enzymes have a common name as well as a systematic name. Names are based on the nature of the enzyme. Its substrate, end product or the type of reaction it catalyses guides the selection of name. Cellulase is so named because it acts on cellulose. Similarly pectinase gets its name from its substrate pectin. Most enzymes have names ending with - 'ase'. Urease, lipase, pectinase and cellulase are examples of enzymes whose names have been coined by suffixing 'ase' to their respective substrates. Trypsin, renin, lysozyme and pepsin, however, are "exceptions that prove the rule."

8

ENZYMES EVERYWHERE

UNKNOWN TO KNOWN

9

Once order was restored to the hitherto chaotic field of enzyme nomenclature, scientists shifted their attention to another quest. This was the sea.rch to discover the nature of enzymes. The search ended in 1926, when James B. Sumner crystallized urease heralding in subsequent reports on isolation and purification of other enzymes. Soon, the statement "Enzymes are proteins" became standard textbook material. Although the sweeping statement has come in for scrutiny and criticism in recent times, it seemed factual enough in the early days of enzymology because it so happened that all the enzymes characterised subsequent to urease were proteins. Or else, they were proteins associated with 7~ ChainA

.:f\~!

./2,.~...~ nucleoside Building triphospnate' 'tmits of ~ Released phosphate

RNA-polymerase helps in transcription

MADE FOR EACH OTHER

19

functions in the cell. It recognises a molecular 'start' signal which indicates to it the beginning of a gene. It then 'reads' the gene. Finally it recognises the 'stop' signal which indicates the end of the gene and thus end of transcription for that particular sequence. The DNA sequence that RNA polymerase recognises as a start signal is called 'promoter'. The same or similar promoter sequences exist at the start of most genes so that the same RNA polymerase can be used to transcribe a large number of genes. Only one of the two DNA strands is transcribed and the final mRNA molecule contains a copy of all the information originally present in the DNA molecule. However, it still needs enzymatic processing or modification before the functional finished products are released. In the early days of molecular biology it was thought that the sequence of nuc1eotides transcribed from the gene was completely represented in the mRNA which in turn, was completely translated into the amino acid language of proteins. With better techniques for gene analysis, this idea has had to be revised .. In higher organisms the genes are found to contain stretches of coding sequences interspersed with pieces of DNA sequences which do not apparently make sense. Such genes are called 'interrupted' or 'split' genes. It is surprising that entire sequences, irrespective of whether 'junk' or not, are copied during transcription. The meaningless sequences that occur within genes are called 'introns'. The sequences of mRNA coding for them are edited out by cellular enzymes. Such post-transcriptional modifications create a continuous, meaningful mRNA message. The portions retained and which finally translate into the amino acid language of proteins are termed 'exons'. The mRNA that finally contains the entire coded informa-· tion has to undergo further modifications before the second step for protein synthesis can begin. In this post-transcriptional processing, the mRNA is given a 'tail' made of many adenine molecules. This 'poly A: tail is believed to help

20

ENZYMES EVERYWHERE

Exon

Intron

Exon

Intron

Exon

DNA

Full length mRNA

1

1I£_og 10'''''''

Processed mRNA

1

TMo,'ation

Protein

Split genes need tailoring before functional enzymes can be made

transfer the mRNA from the nucleus where it is formed tp the cytoplasm where it carries out its function. The 'tail' is sub'sequently removed before the second step in protein synthe.sis can begin. It is called translation, because it involves a change from the nucleotide language of mRNA to the amino acid language of proteins. The mRNA transcript is the intermediary through which the genetic code is conveyed to the protein synthesising machinery of the cell for decoding. In this process, the mRNA gets associated with ribosomes which are compact cellular organelles with their own RNA (ribosomal or rRNA) and a defined set of ribosomal proteins. Another kind of RNA

MADE FOR EACH OTHER

21

Anticodon tRNA has a clover-leaf structure. Code words for amino acids (inset)

called transfer RNA (tRNA) also plays an important role in translation and is considered to be the I adapter molecule' that translates the genetic code into amino acid language. Each mRNA strand becomes associated with ribosomes to form a polyribosome or polysome. The ribosomes associate with the mRNA at a specific end (5' end) and read the

22

ENZYMES EVERYWHERE

message, triplet by triplet. Since the mRNA coded language is written as a series of triplet bases or 'words' it is evident that the nature of the message would change if the reading frame of the code shifted by even a single ba(5e.The first triplet base or codon is called the initiation codon and its spelling is always AVe. It codes for the amino acid methionine. All newly formed protein chains begin with methionine which is later enzymatically cleaved. Each subsequent codon pairs with the complementary 'anticodon' of fhe tRNA and a ,checking factor' ensures that there is a correct fit between the codon and the anticodon. The sequence of codons on an mRNA molecule determines the sequence of anticodons of the "different tRNA molecules, and thus, the different amino acids. There are as many kinds of transfer RNAs in the cell as there are codons. In all there are 61 of them, since these codons are nonsense ones that do not code for any amino acids. The nonsense codons are VAG, VGA and VAA and each of the codons stands for a molecular 'stop' sign indicating the end of translation. Sometimes there may be more than one tRNA for a partkular amino acid. Since the triplet codons of mRNA which are ultimately translated into proteins are dependent on the DNA molecule, it is evident that the DNA molecule holds the key to protein structure. In fact, the central dogma of molecular biology sums it up in one succint sentence. DNA transcription RNA translation protein. Grammatically stated it would read: DNA makes RNA and RNA makes protein. Once the newly formed polypeptide chain is released it ~s folded into the structurally stable molecular conformation. The molecule thus formed is subjected to other modifications as well and only then it is transported to a location where its activity is required by the body. The entire system by which coded genetic information is processed, is replete with scope for error. However, the 'proof-reading' enzymes and 'repair' enzymes ensure that errors are detected and elimin~ted by

23

MADE FOR EACH OTHER

Replication

r@ ~

~)

Translation ~~~ Protein RNA

DNA

Original dogma,

.~

Intron

Transcription

~

processlngZT' L::::::t> m~A~ranSCriPtio~

Reverse Transcription Pre-mRNA