Enzymology Primer for Recombinant DNA Technology

Hyone-Myong Eun

• Publisher: Academic Press
• Year: 1996
• Print ISBN: 9780122437403
• eBook ISBN: 9780080531137
• Topic: Biological Sciences
• Subtopic: Biochemistry
• Index: Biological Sciences

Enzymes are indispensable tools in recombinant DNA technology and genetic engineering. This book not only provides information for enzymologists, but does so in a manner that will also aid nonenymologists in making proper use of these biocatalysts in their research. The Enzymology Primer for Recombinant DNA Technology includes information not usually found in the brief descriptions given in most books on recombinant DNA methodology and gene cloning. - Provides essential basics as well as up-to-date information on enzymes most commonly used in recombinant DNA technology - Presents information in an easily accessible format to serve as a quick reference source - Leads to a better understanding of the role of biocatalysts in recombinant DNA techniques

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Enzymes and Nucleic Acids

General Principles

I. STRUCTURE AND FUNCTION OF ENZYMES

Enzymes are bioreactors that run all biochemical reactions of a living cell in a steady, controlled manner with extraordinary efficiency and specificity. Efficiency means high and widely ranging levels of rate acceleration; specificity means a specific molecular recognition between enzymes and their substrates. The catalytic function is harnessed in a three-dimensional (3-D) protein structure that is simple yet sophisticated and stable yet dynamic. Physiologically, an enzyme is an essential, life-sustaining biocatalyst. Many chemical and biochemical reactions in vitro require enzymes to proceed at reasonable rates under mild conditions of temperature, pH, and in aqueous solutions. Enzymes are thus an indispensable tool not only in recombinant DNA technology, but also in many other areas related to biochemical conversions. As a result, enzymology continues to expand and enzymological techniques are used by workers in a number of disciplines.

By a classical definition, an enzyme is a protein endowed with catalytic functions. As a catalyst, it can only promote a reaction in a thermodynamically favorable direction–it does not change the direction of a reaction. Certain RNA species have been found to possess catalytic functions either alone or in combination with tightly binding protein components. Such catalytic RNAs are referred to as ribozymes. Ribozymes are an exciting new category of enzymes (see Section II,D,4 in this chapter) making an impact on recombinant DNA technology (see Section I,B Chapter 7).

Despite the time-honored recognition of enzymes and their catalytic abilities, the detailed mechanism of enzymatic catalysis has been studied in only a few cases. However, our understanding of how an enzyme works is now entering a new era ushered in by burgeoning recombinant DNA technology. Protein and genetic engineering techniques are now applied to modify and redesign enzymes not only to decipher their structure–function relationships, but also to create novel substrate and reaction specificities and to improve stability and catalytic efficiency. The application of enzymatic principles to the production of antibody has given rise to enzyme-mimicking catalytic antibodies called abzymes. The abzymes, which for the time being “stand at the crossroads of chemistry and immunology” (1), are potentially valuable enzymological tools having tailor-made reaction and substrate specificities. Understanding the intricate inner workings of enzymes offers opportunities to discover novel-concept enzymes and to find innovative and broader uses for biocatalysts.

This volume focuses on the “classical” enzymes only, simply because they are the present-day workhorses in recombinant DNA technology. Starting from some molecular biological and genetic aspects of protein biosynthesis, we will briefly review the fundamental concepts of enzymology–the physicochemical bases of protein structure and catalytic function. This chapter also provides a background for relating the fundamental knowledge of protein biosynthesis, structure, and function to recombinant DNA technology.

A. Biosynthesis of Proteins

1. FLOW OF GENETIC INFORMATION

Proteins are translational products of the genetic information encoded in nucleotide sequences of RNA and/or DNA. The genetic code specifying each amino acid consists of three nucleotide units called codons. Among the possible 64 codons arising from the combination of four natural nucleotides, 61 codons specify amino acids, and the remaining 3 codons code for translation “stop” (Table 1.1). Since there are only 20 natural amino acids used as building blocks for proteins, many amino acids are specified by more than one codon, a phenomenon known as codon degeneracy.

TABLE 1.1

The Genetic Code^a

^aNotable exceptions to the “universal” genetic code include UGA (stop) for Trp in vertebrate mitochondria, UAA (stop) and UAG (stop) for Gln in ciliated protozoa, CUN (leu) for Thr in yeast mitochondria, and AUA (Ile) for Met in vertebrate mitochondria. In some bacterial and mammalian genes, the UGA (stop) codon is used in part for selenocysteine (SeCys).

^bCodes for fMet if in the initiator position.

Conventionally, the DNA sequences that code for functional RNA chains or proteins are termed genes. The RNA that carries the message for translation into a protein is called a messenger RNA (mRNA) and is derived from DNA by a process called transcription. This flow of information from DNA to RNA to protein was once the “central dogma” of molecular biology, but it is now known that the information can also flow in the reverse direction from RNA to DNA, a process called reverse transcription. Among various biological systems, viruses constitute the simplest one which displays diverse modes of genetic information flow. For example, DNA viruses usually express their genes according to the DNA–RNA–protein pathway, whereas some RNA viruses (retroviruses) first reverse-transcribe their genomic RNA into DNA and then follow the “central dogma.” Certain other RNA viruses replicate through an RNA intermediate of opposite sense and do not involve a message conversion to or from DNA at all. Within this group of RNA viruses, some have positive-sense genomic RNAs that can be directly translated into proteins, whereas others have negative-sense genomic RNAs that cannot be directly translated.

2. PROTEIN BIOSYNTHESIS

Protein biosynthesis occurs in ribosomes, the cellular assembly factory where amino acids are covalently linked into a polypeptide chain along the template mRNA. The amino acids are transported into the ribosome in an activated state via coupling to specific carriers known as transfer RNAs (tRNA). The selection of specific amino acids from the cellular pool of amino acids (Tables 1.2 and 1.3) and the task of “charging” to cognate tRNAs are performed by specific aminoacyl-tRNA synthetases. Transfer RNAs contain anticodons, the nucleotide triplets serving as the counterpart of codons. The repertoire of anticodons is substantially smaller than that of codons. This apparent discrepancy is resolved by the fact that anticodons often contain, especially at the first (5′ end) base known as wobble base, noncomplementary bases or inosine which allow nonstandard (or wobble) base pairing with codons. Thus codon degeneracy in mRNA is adequately matched by anticodon wobbling in tRNA. The general process of protein biosynthesis can be divided into three phases of translation: initiation, elongation, and termination.

TABLE 1.2

Common Amino Acids

TABLE 1.3

One-Letter Notation of Amino Acids^a

^aAs recommended by the lUPAC-IUB Commission on Biochemical Nomenclature [BJ (1969) 113, 1–4].

a.: Initiation of translation

i.: Translation in prokaryotes. The start of a prokaryotic mRNA translatio...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• Dedication
• Preface
• Acknowledgments
• List of Abbreviations and Symbols
• List of Journal Abbreviations
• Chapter 1: Enzymes and Nucleic Acids: General Principles
• Chapter 2: Ligases
• Chapter 3: Nucleases
• Chapter 4: Restriction Endonucleases and Modification Methylases
• Chapter 5: Phosphatases and Polynucleotide Kinase
• Chapter 6: DNA Polymerases
• Chapter 7: RNA Polymerases
• Chapter 8: Marker/Reporter Enzymes
• Appendix A: Important Molecular Biological Methods
• Appendix B: Genotypes of Escherichia coli Strains
• Appendix C: Practical Guide for Enzyme Handling
• Index