Protein as enzymes

All enzyme molecules possess the primary, secondary, and tertiary structural characteristics of proteins (see Chapter 20). In addition, most enzymes also exhibit the quaternary level of structure. The primary structure, the linear sequence of amino adds linked through their a-carboxyl and a-amino groups by peptide bonds, is specific for each type of enzyme molecule. Each polypeptide cham is coiled up into three-dimensional secondary and tertiary levels of structure. Secondary structure refers to the conformation of limited segments of the polypeptide chain, namely a-helices, P-pleated sheets, random coils, and p-turns. The arrangement of secondary structural elements and amino acid side chain interactions that define the three-dimensional structure of the folded protein is referred to as its tertiary structure. In many cases biological activity, such as the catalytic activity of enzymes, requires two or more folded polypeptide chains (subunits) to associate to form a functional molecule. The arrangement of these subunits defines the quaternary structure. The subunits may be copies of the 

TABLE 8-1 Enzyme Commission (EC) Numbers, Systenuitic and Trivial Names, Together With Frequently Adopted 

EC Number Systematic Name Trivial Name Abbreviatic 

42 Threo-Ds- isocitrate NAD(P) oxidoreductase (decarhoxylating) Isocitrate dehydrogenase ICD 

2 (5-Glutamyl)-peptide amino-acid 5t A , -glutamyltransferase y-Glutamyltransferase GGT 

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This section begins with a discussion of enzyme nomenclature and is followed with discussions of enzymes as proteins and catalysts. 

The immunoglobulins have been extensively studied by ITP (in serum and csf see Section 2.4.4) and in particular the subclasses of IgG have been studied (H8, HIO, Hll, Z3). An extension of this work has been the demonstration of soluble immune complex formation in vitro (H9), which has obvious implications, particularly for the assessment of immune complex diseases. Preparative work has involved the isolation of, for example, antibodies to pig lactate dehydrogenase (B21, PI) and IgD myeloma protein (Jl). ITP has also been applied in the field of en-zymology, not for the direct measurement of enzymes as proteins, but for the determination of enzyme reaction substrates and products, and hence has been of use in enzyme kinetics. This work is summarized in Table 1. 

It follows that the reactions necessary for life mnst proceed under conditions vastly milder than those considered in chemistry to be mild. As virtually all the reactions we are concerned with are extremely slow under such mild conditions—for practical purposes most of them do not proceed at all—they reqnire catalysts, which in living systems are, as noted a little earlier, enzymes. Until a few exceptions were discovered in recent years they were all believed to be made mainly or entirely of protein. For most biochemical purposes, and specifically for those of this book, we will not go far wrong if we continne to think of enzymes as proteins. 

Enzyme demand in detergent formulations is rising worldwide. The enzyme types used in detergents are proteases, lipases, amylases, and cellulases. New enzymes with unique properties are already being applied to replace the cleaning power of chlorine, while others are being developed for bleaching applications. These enzymes, as protein molecules, would be subjected to harsh con-... 

A large part of our fundamental knowledge about enzymes as proteins and of their behavior as chemical entities was derived from studies on proteolytic enzymes. Classical studies by Northrop and his collaborators ... 

Two-Phase Aqueous Extraction. Liquid—Hquid extraction usually involves an aqueous phase and an organic phase, but systems having two or more aqueous phases can also be formed from solutions of mutually incompatible polymers such as poly(ethylene glycol) (PEG) or dextran. A system having as many as 18 aqueous phases in equiHbrium has been demonstrated (93). Two-phase aqueous extraction, particularly useful in purifying biological species such as proteins (qv) and enzymes, can also be carried out in combination with fermentation (qv) so that the fermentation product is extracted as it is formed (94). 

Several aspects affect the extent and character of taste and smell. People differ considerably in sensitivity and appreciation of smell and taste, and there is lack of a common language to describe smell and taste experiences. A hereditary or genetic factor may cause a variation between individual reactions, eg, phenylthiourea causes a bitter taste sensation which may not be perceptible to certain people whose general abiUty to distinguish other tastes is not noticeably impaired (17). The variation of pH in saUva, which acts as a buffer and the charge carrier for the depolarization of the taste cell, may influence the perception of acidity differently in people (15,18). Enzymes in saUva can cause rapid chemical changes in basic food ingredients, such as proteins and carbohydrates, with variable effects on the individual. 

As described earlier, translation of the EPSPS mRNA of plants results in the formation of a protein which has an AJ-terminal extension. The AJ-terminal extension, referred to as the chloroplast transit peptide, is necessary and sufficient for the import of the preprotein by the chloroplast. Once imported by the chloroplast, the transit peptide is cleaved releasing the mature enzyme. As expected, introduction of the EPSPS transit peptide to other protein sequences results in the importation of the fusion protein by the chloroplast. 

This chapter lists some representative examples of biochemicals and their origins, a brief indication of key techniques used in their purification, and literature references where further details may be found. Simpler low molecular weight compounds, particularly those that may have been prepared by chemical syntheses, e.g. acetic acid, glycine, will be found in Chapter 4. Only a small number of enzymes and proteins are included because of space limitations. The purification of some of the ones that have been included has been described only briefly. The reader is referred to comprehensive texts such as the Methods Enzymol (Academic Press) series which currently runs to more than 344 volumes and The Enzymes (3rd Edn, Academic Press) which runs to 22 volumes for methods of preparation and purification of proteins and enzymes. Leading referenees on proteins will be found in Advances in Protein Chemistry (59 volumes. Academic Press) and on enzymes will be found in Advances in Enzymology (72 volumes, then became Advances in Enzymology and Related Area of Molecular Biology, J Wiley Sons). The Annual Review of Biochemistry (Annual Review Inc. Patio Alto California) also is an excellent source of key references to the up-to-date information on known and new natural compounds, from small molecules, e.g. enzyme cofactors to proteins and nucleic acids. 

The most frequent of the domain structures are the alpha/beta (a/P) domains, which consist of a central parallel or mixed P sheet surrounded by a helices. All the glycolytic enzymes are a/p structures as are many other enzymes as well as proteins that bind and transport metabolites. In a/p domains, binding crevices are formed by loop regions. These regions do not contribute to the structural stability of the fold but participate in binding and catalytic action. 

For many years hemoglobin was the only allosteric protein whose stereochemical mechanism was understood in detail. However, more recently detailed structural information has been obtained for both the R and the T states of several enzymes as well as one genetic repressor system, the trp-repressor, described in Chapter 8. We will here examine the structural differences between the R and the T states of a key enzyme in the glycolytic pathway, phosphofructokinase. 

Enzyme A protein that funetions as a bioeatalyst in a ehemieal reaetion. Any group of eatalytie proteins that are produeed by living eells mediating and promoting the ehemieal proeesses of life without... 

The natural polymers known as proteins make up about 15% by mass of our bodies. They serve many functions. Fibrous proteins are the main components of hair, muscle, and skin. Other proteins found in body fluids transport oxygen, fats, and other substances needed for metabolism. Still others, such as insulin and vasopressin, are hormones. Enzymes, which catalyze reactions in the body, are chiefly protein. 

Enzymes are protein molecules. While all enzymes are proteins, we do not imply that all proteins can act as enzymes. The protein molecules of enzymes are very large, with molecular weights of the order of 100,000. In contrast, the substance upon which the enzyme acts (called a substrate) is very small in comparison with the enzyme. This creates a picture of the reaction in which the small substrate molecule becomes attached to the surface of the large protein molecule, at which point the reaction occurs. The products of the reaction then dissociate from the enzyme surface and a new substrate molecule attaches to the enzyme and the reaction is repeated. We can write the following sequence ... 

In general, enzymes are proteins and cany charges the perfect assumption for enzyme reactions would be multiple active sites for binding substrates with a strong affinity to hold on to substrate. In an enzyme mechanism, the second substrate molecule can bind to the enzyme as well, which is based on the free sites available in the dimensional structure of the enzyme. Sometimes large amounts of substrate cause the enzyme-catalysed reaction to diminish such a phenomenon is known as inhibition. It is good to concentrate on reaction mechanisms and define how the enzyme reaction may proceed in the presence of two different substrates. The reaction mechanisms with rate constants are defined as ... 

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