Structure enzyme

Enzymes are macromolecular protein catalysts, consisting of linear condensed polymers of a-amino acids joined in amide linkages. Each enzyme has a genetically determined and unique primary sequence, and it folds in three dimensions into a precise orientation (Lodish et al, 1999). 

The primary structure of a protein is specified by the order in which the amino acids are linked together through the peptide bonds. The most important feature of this structure is the peptide linkage shown in Fig. 1. 

Because of resonance, which gives the N-C bond a partially double bond character, the peptide bond is planar. Furthermore, the a-carbons are almost always trans. These two features of the peptide bond play a dominant role in determining protein structure. The other covalent linkage of importance is the disulfide bond that joins different parts of the protein chain in enzymes secreted outside the cells. Special note should also be made of an imino acid proline, which creates a very rigid peptide bond. 

The final stmcture of a protein is determined by the above factors and optimization of noncovalent interactions involving the peptide backbone and the amino acid side chains. The large variety of amino acid side chains provides the possibility of several different types of non-covalent interactions, such as van der Waals, jt-electron stacking, hydrogen bonding, and electrostatic. The main types of amino acid side chains and their functions are summarized in Table 1. 

While the static aspects of electrostatic interactions can be readily formulated, the dynamics are more difficult to ascertain (Hammes, 1982). A few kinetic studies for ion-pair formation have been carried out, and the rate constants appear to be those of diffusion-controlled reactions. For reactions between small molecules with univalent charges of opposite sign, fcf, the rate constant for 

Proteins, with a specific function and isolated from a single source, usually have a homogeneous population of molecules all with the same unique amino acid sequence. Yet with 20 different amino acids possible at each position in a polypeptide chain of n residues, 20 different primary structures are theoretically possible. Furthermore, the great majority of all molecules of a natural protein may exist in a unique conformation despite the degrees of freedom formally permitted by rotation about the peptide backbone (motility) and side chains (mobility). For example, with only 3 conformations defined per residue, a polypeptide chain of 210 residues would have a theoretical possibility of existing in 10 °° different conformations. 

Reversible unfolding of proteins has been known for some time. The folding of several proteins occurs spontaneously showing that the required information for folding is present in the protein s primary structure [1]. 

The production of a disordered polypeptide chain by removing just a few of the interactions which normally contribute to the stability of the folded state is well illustrated by breaking the 4 disulphide bonds in ribonuclease A - even in the absence of a denaturant, the reduced protein is fully unfolded despite all other favourable interactions, such as hydrogen bonds and hydrophobic forces, still being possible [2]. 

Detailed models of the folded states of proteins depend almost entirely on X-ray diffraction analysis of the protein crystal. Although side chains and flexible loops on the surface may be mobile in solution, protein conformation in solution is essentially that determined in the solid crystal. The atoms of folded proteins are generally well fixed in space. 

The overall structure-of a folded protein is remarkably compact. The extended chain of carboxypeptidase, with 307 residues, would be 1114 A long but the maximum dimension of the folded molecule is only about 50 A. However, the polypeptide topology in roughly spherical, globular proteins has never been observed to form knots [3]. 

Several factors have contributed to elucidating enzymological details of degradative pathways  

Advances in molecular biology have led to the construction of strains that include the relevant degradative genes. Amplihcation of these, and expression of them in Escherichia coli has made possible the isolation of pure enzymes in quantities suitable for crystallization. 

There has been remarkable progress in techniques for crystallizing proteins. 

Advances in the technology of x-ray diffraction have made it possible to achieve three-dimensional structures of enzymes together bound, in many cases, to their substrates. 

Albert K (1995) On-line nse of NMR detection in separation chemistry. J Chromatogr A 703 123. 

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Fersht, A. Enzyme Structure and Mechanism, 2nd ed. New York W.H. Freeman, 1984. 

James, M.N.G. An x-ray crystallographic approach to enzyme structure and function. Can. J. Biochem. 

T. C. Bruice and S. I Benkovic, Bioorganic Mechanisms, Vol. 1, W. A. Benjamin, New brk, 1966, pp. 1-258 W. P. Jencks, Catalysis in Chemistry and Enzymology, McGraw-Hill, New York, 1969 M. L. Bender, Mechanisms of Homogeneous Catalysis from Protons to Proteins, Wiley-Interscience, New York, 1971 C. Walsh, Enzymatic Reaction Mechanisms, W. H. Freeman, San Francisco, 1979 A. Fersht, Enzyme Structure and Mechanism, 2nd ed., W. H. Freeman, New York, 1985. 

A. Eersht, Enzyme structure and mechanism, W. H. Ereeman and Company, New York 1985. 

Conti, E., Franks, N. P., and Brick, P. (1996). Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes. Structure 4 287-298. 

In the enzyme design approach, as discussed in the first part of this chapter, one attempts to utilize the mechanistic understanding of chemical reactions and enzyme structure to create a new catalyst. This approach represents a largely academic research field aiming at fundamental understanding of biocatalysis. Indeed, the invention of functional artificial enzymes can be considered to be the ultimate test for any theory on enzyme mechanisms. Most artificial enzymes, to date, do not fulfill the conditions of catalytic efficiency and price per unit necessary for industrial applications. 

Fersht, A. 1985 Enzyme structure and mechanism. New York W. H. Freeman. 

Despite the wide diversity of enzyme structures, most enzyme activity follows a general mechanism that has several reversible steps. In the first step, a reactant molecule known as a substrate (S) binds to a specific location on the enzyme (E), usually a groove or a pocket on the surface of the protein E + S ES The substrate binds to the active site through intermolecular interactions that usually include significant amounts of hydrogen bonding. 

Schroeder, W. A. "Separation of Peptides by Chromatography on Columns of Dowex 1 with Volatile Developers", In "Methods In Enzymology", p. 214, Vol. XXV, "Enzyme Structure, Part B", C. H. W. Hlrs and S. N. Tlmasheff, Editors, Academic Press, New York, 1972. 

Recently, the distribution of 2,3-dihydroxybenzoate decarboxylase has been found in a variety of fungal strains (unpubhshed data), and the carboxylation activity for catechol is confirmed by the reaction using resting cells (or cell-free extract) in the presence of 3M KHCO3. The detailed comparative studies of enzyme structures and catalytic properties between 2,3-dihydroxybenzoate decarboxylase and 3,4-dihyroxybenzoate decarboxylase might explain how the decarboxylases catalyze the regioselective carboxylation of catechol. 

Apart from mode of action and kinetics of wild type enzymes structure function relationships of these industrially important enzymes is of high interest to provide the necessary knowledge for genetic engineering of desired properties. As a first approach the identification of catalytically important residues was addressed in conjunction with the elucidation of the three dimensional structure [15]. 

The draggability of enzymes as targets reflects the evolution of enzyme structure to efficiently perform catalysis of chemical reactions, as discussed in the following section. 

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