The Protein Nature of Enzymes

All enzymes are proteins and thus are composed of alpha-amino acid residues, arranged in a unique sequence for each protein, each sequence being determined genetically by a complex mechanism which will be considered elsewhere in this book. These amino acid residues are bonded by covalent peptide bonds, formed by a condensation pro- 

Nucleophilic substitution-saturated systems (occurs readily only if the carbon is attached to an atom with a positive charge) 

Nucleophilic—reactions of carboxylic acid derivatives (attack of a base on an electrophilic carbon atom) 

Addition and elimination involving carbon-carbon (enolate anions) a. Dehydration of alcohols—enoyl hydratase, fumarase, aconitase 

Addition to carbonyl groups (usually as a step in a process resulting in hemiacetal, or carbanol-amine formation) 

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Laufberger had tried to obtain the protein from horse liver, but it did not crystallize, and as he described to me when I met him in Prague some years ago, in those days everyone wanted to have protein crystals as a criteria of purity. Although James Sumner had crystallized jack bean urease in 1926, his preparations were somewhat impure, and it was only in the mid-1930s, when John Northrop and Moses Kubnitz showed that there is a direct correlation between the enzymatic activities of crystalline pepsin, trypsin and chymotrypsin that the protein nature of enzymes was generally accepted. 

The protein nature of enzymes was established through the seminal work of James Sumner. In 1926, Sumner succeeded in isolating the enzyme urease in crystalline form from jack bean meal. This was the first time in history that an enzyme had been obtained in crystalline, though not completely pure, form. Subsequently, Sumner established that the crystalline enzyme was a protein. Urease is an enzyme that degrades one of the human end products of nitrogen metabolism, urea, to ammonia and carbon dioxide ... 

Like many classic contributions to science, this spectacular advance was initially met with criticism and even derision. However, Sumner was not deterred he took his show on the road and, through demonstration, convinced many scientists of the protein nature of enzymes. Subsequently, a number of enzymes were crystallized in the 1930s by John Northrup and Moses Kunitz and were also shown to be proteins. These later preparations were essentially pure. Northrup and Kunitz demonstrated that enzyme activity paralleled the amount of protein present, laying the issue to rest. Sumner and Northrup shared the 1946 Nobel Prize in Chemistry. ... 

The second aspect refers to the protein nature of enzymes. In 1894 Fischer (Fischer, 1909) stated that amongst the agents which serve the living cell the proteins are the most important. He was convinced that enzymes are proteins. The role of this key problem may be illustrated with a citation from Fruton (1979) ... the peptide theory was indeed only a hypothesis fifty years after Franz Hofmeister and Emil Fischer advanced it... (in 1902). The nature and stracture of proteins remained unknown throughout the 19th century remarkably, technological applications were nevertheless put into practice since the middle of the century (see above), based on their action, eventually recognized as catalysis, only. 

In 1926, James Sumner established the protein nature of enzyme. He was responsible for the isolation and crystallization of urease, which provided a breakthrough in studies of the properties of specific enzymes. 

Their lability to heat is one indication of the protein nature of enzymes, Another is their sensitivity to pH. All enzymes have one pH at which they work fastest, and slight shifts in pH away from this point bring sharp falls in catalytic activity. A typical pH curve for an enzyme is that of Figure 12, and it is plain that this effect is... 

The proteinic nature of enzymes means that they have a large number of reactive groups in common and can be simultaneously immobilized... 

A second kind of evidence of the protein nature of these enzymes is afforded by our studies of the effects of antiseptics. Here it was found that the amylases are enormously more sensitive to antiseptics of the type which precipitate proteins (e.g., cupric sulfate) than to those of the lipoiddissolving type (e.g., toluene).6... 

The chemical nature of enzyme was controversial for a long time, until Buchner succeeded in isolating an enzyme system (zymase) from yeast in a cell-free extract in 1897.2) Urease was then crystallized by Sumner in 1926,3) followed by crystallization of several proteolytic enzymes by Northlop and his colleagues. At present the chemical nature of enzyme is defined as a protein with catalytic activity based on the specific activaiton of its substrate. However, this definition has been somewhat open to debate since a catalytic RNA, ribozyme, was discoved in 1982. 

The purification and properties of porcine pancreatic lipase have been discussed in a recent review (142). Only the most significant facts related to the protein nature of the enzyme will therefore be presented here. 

For enzyme-based biosensors the mode of detection is based on the catalytic activity and/or binding capacity. Because of the protein nature of almost all enzymes, the catalytic activity depends on the conformation. Exceptions are catalytic ribonucleic acids called DNA biosensors or genosensors. DNA fragments are used as probes for detecting low concentrations of DNA in large samples (see also Part I, Chapters 2 and 3). Because of the highly diluted DNA concentration, microelectromechanical systems which are able of performing PCRs are employed. 

Enzymes that hydrolyze proteins and other compounds composed of amino acids were among the first biological catalysts to be discovered, and they have continued to be prominent in studies of enzyme structure, kinetics, activation, and mechanism of action. The crystallization of the enzyme urease by Sumner was followed by the crystallization of various proteolytic enzymes in the laboratory of Northrop. These studies established that catalytic activity is associated with what appear to be pure proteins all well-defined enzymes isolated subsequently have also proved to be proteins, although many contain additional components. The study of enzymatic reactions involving proteins as substrate, therefore, gives insight into the chemical nature of enzymes as well as the mechanisms by which they act. 

From almond emulsin, enzymes with a jS-glucosidase value as high as 16 have been prepared. (The ordinary almond emulsins have a value of 1 or less.) The properties of such preparations have been studied by Helferich and associates (16). The elementary composition agrees with that of protein substances, but the hydrogen content is somewhat greater than usual. The protein nature of the material also agrees with the action of the anions of neutral salts on the enzymic activity since the order of activation by... 

Enzymes are globular proteins with greatly differing particle sizes (cf. Table 1.26). As outhned in section 1.4.2, the protein structure is determined by its amino acid sequences and by its conformation, both secondary and tertiary, derived from this sequence. Larger enzyme molecules often consist of two or more peptide chains (subunits or protomers, cf. Table 1.26) arranged into a specified quaternary structure (cf. 1.4.2.3). Section 2.4.1 will show that the three dimensional shape of the enzyme molecule is actually responsible for its specificity and its effective role as a catalyst. On the other hand, the protein nature of the enzyme restricts its activity to a relatively narrow pH range (for pH optima, cf. 2.5.3) and heat treatment leads readily to loss of activity by denaturation (cf. 1.4.2.4 and 2.S.4.4). 

Thomas, P.G., Russel, A.J., Fersht, A. Tailoring the pH dependence of enzyme catalysis using protein engineering. Nature 318 375-376, 1985. 

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