Enzyme-Based Organic Synthesis. Cheanyeh Cheng

technology and corresponding instrumentation, hundreds of enzymes had been purified and discovered in the middle of nineteenth century. Nevertheless, the sequencing of proteins and enzymes was not until the work of William H. Stein et al [8]. who first complete the sequence of ribonuclease A (an enzyme with only 124 amino acids) in 1960. Later, in 1972, William H. Stein and Stanford Moore shared the Nobel Prize in chemistry.

1.2 Enzyme Structure and Catalytic Function

      1.2.1 Enzyme Structure

      With the exception of a small group of catalytic RNA molecules, all enzymes are protein. The protein nature of enzyme has been elucidated about a century ago that has led fast and broad progress in chemistry, biochemistry, and biology, in addition, led the development of many new fields such as enzymology, bioorganic chemistry, and molecular biology. In order to understand enzyme and how its function as a catalyst, one must know the enzyme structure first. Since enzyme is a kind of protein, its structure follows the four‐level structure of protein, namely, the primary structure, the secondary structure, the tertiary structure, and the quaternary structure.

      Protein is a polymer of amino acids that is referred to as peptides or proteins. Peptides are chains of amino acids joined through a substituted amide linkage, termed a peptide bond, which is formed by dehydration to remove the elements of water from the α‐carboxyl group of one amino acid and the α‐amino group of another. When a few amino acids (usually, less than 10) are joined in this fashion, the structure is called an oligopeptide. When many amino acids are joined, the product is called a polypeptide. Proteins that may have thousands of amino acid residues are polypeptides. Therefore, “protein” and “polypeptide” are sometimes used interchangeably. However, molecules with molecular weight below 10 000 are generally, referred to as polypeptides. In a peptide, the amino acid residue at one end with a free α‐amino group is the amino‐terminal (or N‐terminal) residue, while at the other end, the residue with a free carboxyl group is the carboxyl‐terminal (C‐terminal) residue [9].

Since proteins are large macromolecules, the complexity of their 3D structure cannot be described easily like small molecules. Therefore, four levels of structure are used to define the complete 3D structure of protein. The primary structure is the amino acid sequence of a polypeptide chain that describes the order of all covalent bonds, mainly peptide bonds and disulfide bonds, linking amino acid residue in the polypeptide as illustrated in Scheme 1.1. The importance of primary structure is in determining the secondary, tertiary, quaternary structures of proteins, and thus their biological functions, which can be demonstrated by the hereditary disease sickle‐cell anemia of human.

Scheme 1.1 The primary structure of a polypeptide chain linked by the peptide bond shows the sequence of amino acids.

      Part of the very long chain polypeptide can be coiled or folded into units by amino acid residues within a short distance to form recurring structural patterns of secondary structure such as the α‐helix of α‐keratin. The helix is a part of the tertiary structure that is the overall 3D arrangement or folding of a polypeptide. An example of the tertiary structure is myoglobin, a globular protein with 153 amino acid residues. The secondary structure refers to the spatial arrangement of amino acid residues that are adjacent in the primary structure, whereas tertiary structure includes longer‐range aspects of the primary structure. When a protein has two or more polypeptide subunits that are associated with each other or one another, their arrangement in space is referred to as quaternary structure. Hemoglobin consisting of four polypeptide subunits is the most well‐known protein with a complex quaternary structure.

      1.2.2 Catalytic Function

      The folding of long polypeptide chain to form tertiary or quaternary structure is caused by chemical or physical forces such as disulfide linkage, hydrogen bonding, acid–base interaction (salt bridge), and hydrophobic interaction. Folding of polypeptides into two or more stable globular units is called domains. Different domains often play distinct functions, such as binding molecules or interaction with other proteins. A molecule bound reversibly by a protein is called ligand. The site on the protein that binds the ligand is called the binding site. When a protein binds a ligand, the 3D structure of protein is often caused by a conformational change to permit a tighter binding to the ligand. This kind of binding with structural adaption between protein and ligand is called induced fit mechanism. Enzymes have catalytic function that binds and chemically rapidly transforms other molecules. For enzyme‐catalyzed reaction, the molecule bound and acted by the enzyme is called substrate; the binding site is called the active site or catalytic site.

Enzyme is an efficient catalyst and is responsible for thousands integrated chemical reactions of the biological process occurred in the living system. Just like the usual inorganic catalyst, enzymes catalyze a reaction by lowering the transition state energy (the activation energy) of the activated complex and by raising the ground state energy. On the other hand, the catalysis of enzymes, not like simple inorganic catalysts, proceeds by forming several transition states and each with low activation energy instead of one activated complex of greater activation energy. The rate of enzyme‐catalyzed reaction for a simple one enzyme, one substrate and one product system with the following mechanism was studied by Michaelis and Menten in 1913 (Scheme 1.2). In this mechanism, enzyme (E) binds the substrate (S) to form an enzyme–substrate (ES) complex and subsequently the ES breaks down to the product (P).

Scheme 1.2 The proposed enzyme reaction mechanism by Michaelis and Menten.

According to this mechanism, the enzyme‐catalyzed reaction rate equation called Michaelis–Menten equation (Eq. 1.1) was derived by Michaelis and Menten with the second step as the rate‐limiting step and derived by Briggs and Haldane using steady‐state assumption. The term V ₀ is the initial rate, V _max is the maximum reaction rate, [S] is the substrate concentration, and K_m is the Michaelis constant.

(1.1)

For a more common case, enzyme product complexes (EP) release, EP → E + P, is the rate‐limiting step, which is described as the reaction Scheme 1.3.

Therefore, a more general rate constant called the turnover number, k cat, is defined to describe the limiting rate of any enzyme‐catalyzed reaction at saturation, that is, V _max = k _cat[E _t]. In this situation, the Michaelis–Menten equation becomes Eq. 1.2

(1.2)

The turnover numbers of several enzymes are given in Table 1.1.

Скачать книгу