Aspects of Enzyme Chemistry

It is these aspects that we intend to cover here. After an outline of some pertinent aspects of enzyme chemistry, the various enzymes degrading and synthesizing starch are considered in relation to what is known about how each enzyme works. 

Older methods of fractionating protein admixtures in aqueous solution usually involved the stepwise addition of ammonium sulfate, or an organic solvent, such as acetone or ethanol. These methods have been used in the crystallization of several starch-metabolizing en- 

Gel-permeation chromatography [on a modified dextran, poly (acrylamide), or agarose gel] separates proteins on the basis of molecular size, alpha- and hefa-Amylases have been purified on Sephadex, a gel of cross-linked dextran, but anomalous behavior is observed with alpha-amylases on such gels these enzymes are eluted from the gel less readily than would be expected on the basis of their molecular size, and it is thought that a weak, protein—gel complex is formed, similar in nature to an enzyme—substrate complex. 

It is extremely difficult to show that a protein is pure and consists of one component only. Proteins can crystallize while still impure, and so crystallinity alone cannot be taken as proof of homogeneity. Indeed, no single test is available to prove that an enzyme preparation is homogeneous, and several different tests must be applied. 

Many starch-degrading enzymes have been investigated electro-phoretically in attempts to confirm their homogeneity, for example, alpha-amylase, heta-amylase, phosphorylase, and pullulanase.  

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Biocatalysis refers to catalysis by enzymes. The enzyme may be introduced into the reaction in a purified isolated form or as a whole-cell micro-organism. Enzymes are highly complex proteins, typically made up of 100 to 400 amino acid units. The catalytic properties of an enzyme depend on the actual sequence of amino acids, which also determines its three-dimensional structure. In this respect the location of cysteine groups is particularly important since these form stable disulfide linkages, which hold the structure in place. This three-dimensional structure, whilst not directly involved in the catalysis, plays an important role by holding the active site or sites on the enzyme in the correct orientation to act as a catalyst. Some important aspects of enzyme catalysis, relevant to green chemistry, are summarized in Table 4.3. 

Table 4.3 Aspects of enzyme catalysis relevant to green chemistry...

Biomedical analytical chemistry happens to be one of the latest disciplines which essentially embraces the principles and techniques of both analytical chemistry and biochemistry. It has often been known as clinical chemistry . This particular aspect of analytical chemistry has gained significant cognizance in the recent past by virtue of certain important techniques being included very much within its scope of analysis, namely colorimetric assays, enzymic assays, radioimmunoassays and automated methods of clinical analysis. 

In examining the structure of a polysaccharide, it is convenient to consider the methods involved under the three main headings (a) quantitative analysis, (b) methylation, and (c) periodate oxidation. These techniques may be supplemented by partial or enzymic hydrolysis as the circumstances indicate. Each of these aspects of polysaccharide chemistry may be aided by the application of gas-liquid chromatography, either qualitative or quantitative, or both. Thus, separations impossible by other techniques may often be achieved, and analytical data obtained in a fraction of the time demanded by other methods. 

Catalytical aspects of supramolecular chemistry will be discussed in some detail in the next chapter. In this section only cyclodextrins as enzyme models will be briefly presented. Several examples of cyclodextrins catalytic activity have been reported. The acceleration of the reaction rate upon adding of CDs is... 

Zn+2, Mn+2, Fe+2, Cd+2, Co+2, and Ni+2, although diminished activity results (35, 53). Fundamentally the enolase and aconitase reactions are closely related, since the net result of both is the addition or subtraction of a molecule of water. In spite of this similarity, the metal ions associated with these two reactions are very different. It is one of the puzzling aspects of metalloenzyme chemistry that every enzyme has a different metal ion specificity. Each of these enzymes is associated in its natural state with a specific metal ion, which differs from enzyme to enzyme. It is possible to remove the naturally occurring metal from many of these enzymes and to reactivate them by the addition of other metals, as has been shown in the case of carboxypeptidase. The order in which the various metal ions fall in their ability to activate the different enzymes again varies from enzyme to enzyme. 

The chief use of molybdenum is in steels. The oxides and sulfides have some applications as catalysts. Molybdenum is the only element in the second and third transition series which appears to have a major role as a trace metal in enzymes. Several aspects of molybdenum chemistry have been widely studied in order to gain a better understanding of the biological relevance. Molybdenum is one of the few elements which currently has its own series of international conferences.1... 

In addition to interest in this enzyme because of its catalytic characteristics, a considerable body of information has accumulated on staphylococcal nuclease as a protein molecule. Its relatively small size, the absence of covalent cross-linkages, and its behavior upon binding a variety of ligands have made it an ideal model substance for the study of various aspects of protein chemistry including X-ray crystallography. These investigations are reviewed in the present chapter and in Chapter 7 by Cotton and Hazen, this volume, on the three-dimensional structure (19). 

Before we could take up this study in general, we had to solve one of the more bothersome aspects of cyclodextrin chemistry. It was believed strongly that cyclodextrin would bind substrates only in pure water solution, and this was a serious defect. First of all, it severely restricted the range of substrates that could be examined, since many interesting molecules have low water solubility. As a second point, it made it difficult to examine another feature of enzyme-catalyzed reactions. One of the roles that can be ascribed to the large protein mass, which contains the functional groups of an enzyme, is the function of water exclusion. That is, enzyme reactions can be considered to be operating in a nonaqueous or only partially aqueous medium. 

B. Ganem, in Comprehensive Natural Products Chemistry, Vol. 5 Enzymes, Enzyme Mechanisms, Proteins and Aspects of NO Chemistry (Ed. C. D. Poulter), Elsevier, Oxford, UK, 1999, pp. 343. 

Enzyme-catalysed chemical reactions can be up to 10 times faster than their counterparts in organic chemistry. These amazing rate accelerations represent one of the most controversial issues of enzyme catalysis. No truly consistent explanation for this phenomenon has yet been advanced. Attempts to mimic this aspect of enzyme catalysis at specially designed artificial systems are numerous, but generally the interpretation of these results is not free from ambiguities. Nevertheless, it is appropriate to consider at least briefly some approaches illustrative of the general trends of these studies. 

GIO. Guilbault, G. G., Use of enzymes in and kinetic aspects of anal3rtical chemistry. Anal. Chem. 42, 334R-349R (1970). 

The aspects of enzyme-catalyzed reactions cited above are so far beyond what is normally achieved in chemical reactions that there is a tendency to believe that unusual principles must be invoked to explain the results. This is absolutely imtrue just like any other kind of catalysts, enzymes do chemistry, not magic The goal of this and the next section is to illustrate how enzymatic reactions are controlled by familiar chemical principles of structure and reactivity. 

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