Enzymes and Their Structure

Enzymes are specific proteins performing the role of catalyst in living organisms. All enzymes can be subdivided into six classes. Until now the enzymes of two classes, viz. oxidoreductases catalyzing oxidation-reduction reactions and hydrolases promoting hydrolysis reactions, have been used in electrochemical systems. 

Oxidation-reduction reactions in the presence of oxidoreductases occur through the transfer of either electrons or hydrogen atoms. Table 1 shows certain enzymes in the oxidoreductase class, which are finding application in electrochemical research. 

Hydrogenase Glucose oxidase Xanthine oxidase Aldehyde oxidase Alcohol dehydrogenase 

Hydrolases catalyze the hydrolytic splitting of covalent bonds ester, peptide, acid anhydride, etc. Table 2 contains data on hydrolases which have been utilized in research on bioelectrocatalysis. 

An enzyme is a high-molecular weight protein with an active center where the binding of the substrate and its chemical transformation take place. The protein part of the enzyme consists of a-L-amino acids linearly interconnected by peptide bonds. The lower boundary of the molecular weight of proteins is arbitrarily assumed to be 5000. The upper boundary amounts to some hundred thousand or even millions. The structural diversity of proteins is due to a wide choice of amino acids and the presence of several levels of structural organization. 

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Sigman, D., ed. The Enzymes, Yo. 20, Mechanisms of Catalysis. San Diego Academic Press, 1992. [Part of a definitive series on enzymes and their structures and functions.]... 

The enzymes and their structural genes for the three key reactions for the synthesis of PHAs of short-chain-length monomers (three to five carbon atoms) have been extensively studied, and their salient features are summarized below. 

Tungsten-containing enzymes have been found to mediate a variety of reactions both in aerobic and anaerobic bacteria, and their structure may plausibly be assumed to be analogous to the molybdopterins ... 

The study of cellulases has progressed considerably in the present decade. Recombinant DNA techniques have been applied and protein-chemical and structural studies have provided new insights. Crystallization of the first cellulases has succeeded recently and detailed structural information may be expected soon. It is hoped that this will give a further incentive to studying the intricate reaction mechanism of these enzymes and their substrate interactions (adsorptions). The important synergy phenomena certainly need a more systematic approach and new techniques should be applied in this area. 

Lipases belong to the subclass of serine hydrolases, and their structure and reaction mechanism are well understood. Their common a/p-hydrolase enzyme fold is characterized by an a-helix that is connected with a sharp turn, referred to as the nucleophilic elbow, to the middle of a P-sheet array. All lipases possess an identical catalytic triad consisting of an Asp or Gin residue, a His and a nucleophilic Ser [14]. The latter residue is located at the nucleophilic elbow and is found in the middle of the highly conserved Gly—AAl—Ser—AA2—Gly sequence in which amino acids AAl and AA2 can vary. The His residue is spatially located at one side of the Ser residue, whereas at the opposite side of the Ser a negative charge can be stabilized in the so-called oxyanion hole by a series of hydrogen bond interactions. The catalytic mechanism of the class of a/P-hydrolases is briefly discussed below using CALB as a typical example, since this is the most commonly applied lipase in polymerization reactions [15]. 

After receiving his Ph.D., Dr. French spent two years, 1942 to 1944, as a post-doctorate fellow in the laboratories of Professors J. D. Edsall and E. J. Cohn at Harvard Medical School. During this period, French worked in the area of amino acids and proteins, and he became especially interested in relating the structure of amino acids and proteins to chemical reactivity. With Dr. Edsall, he published an excellent review on the reactions of formaldehyde with amino acids and proteins. In this stage of his career, his interest was aroused in proteins that possess enzymic activity. In later years, much of his research was devoted to enzymes and their mode of action, and to the molecular mechanisms and theoretical aspects of enzyme action. 

Smith, S. 1976. Structural and functional relationships of fatty acid synthetases from various tissues and species. In Immunochemistry of Enzymes and Their Antibodies. M.G.J. Salton, (Editor). John Wiley Sons, New York, pp. 125-146. 

There are methods used Lo study enzymes other than those of chemical instrumental analysis, such as chromatography, that have already been mentioned. Many enzymes can be crystallized, and their structure investigated by x-ray or electron diffraction methods. Studies of the kinetics of enzyme-catalyzed reactions often yield useful data, much of this work being based on the Michaelis-Menten treatment. Basic to this approach is the concept (hat the action of enzymes depends upon the formation by the enzyme and substrate molecules of a complex, which has a definite, though transient, existence, and then decomposes into the products, of the reaction. Note that this point of view was the basis of the discussion of the specilicity of the active sites discussed abuve. 

The general scheme of the biosynthesis of catecholamines was first postulated in 1939 (29) and finally confirmed in 1964 (Fig. 2) (30). Although not shown in Figure 2, in some cases the amino acid phenylalanine [63-91-2] can serve as a precursor it is converted in the liver to (-)-tyrosine [60-18-4] by the enzyme phenylalanine hydroxylase. Four enzymes are involved in E formation in the adrenal medulla and certain neurons in the brain tyrosine hydroxylase, dopa decarboxylase (also referred to as L-aromatic amino acid decarboxylase), dopamine-P-hydroxylase, and phenylethanolamine iV-methyltransferase. Neurons that form DA as their transmitter lack the last two of these enzymes, and sympathetic neurons and other neurons in the central nervous system that form NE as a transmitter do not contain phenylethanolamine N-methyl-transferase. The component enzymes and their properties involved in the formation of catecholamines have been purified to homogeneity and their properties examined. The human genes for tyrosine hydroxylase, dopamine- 3-oxidase and dopa decarboxylase, have been cloned (31,32). It is anticipated that further studies on the molecular structure and expression of these enzymes should yield interesting information about their regulation and function. 

There are several recent reviews of the molybdenum and tungsten enzymes [4-6,23,26-36], In this chapter, we first define the metallocofactors and offer a compilation of the enzymes and their diverse activities. We then focus on the active-site structures, highlighting the confluence of crystallographic and spectroscopic studies. This is followed by a discussion of pertainent spectroscopic, structural, reactivity, and theoretical model studies. We then turn our attention to the mechanisms of catalytic activity of the molybdenum and tungsten enzymes. 

What happens to glucose oxidase upon absorption can be understood in terms of the actual structure of an enzyme. Enzymes are relatively complex, and their structures as organic molecules are difficult to draw. However, it is possible to make a representation, although much is lost in the absence of a three-dimensional model. A diagram due to L. Sawyer (1991) of the enzyme p-lactoglobulin is shown in Fig. 1423. 

The detailed mechanism by which AChE and BChE hydrolyze ACh has been the subject of much research, especially since the crystal structure of the Torpedo califomica AChE was elucidated by Sussman et al. in 1991 [12]. (Reviews of these enzymes and their interactions can be found in Refs. [5,13]). This mechanism will be described here only briefly, as an introduction to the reaction of the enzyme with carbamates. The active site of AChE is located at the bottom of a 20 A-deep gorge, where acetylcholine fits in by attachment of the quaternary ammonium group to the so-called anionic site (mainly through cation interaction with the n electrons of Trp84), and by dipole interactions between the ester group and Ser200 at the esteratic site . 

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