Mechanism of Enzyme Action

Introduction - Enzyme Characteristics: The basic mechanism by which enzymes catalyze chemical reactions begins with the binding of the substrate (or substrates) to the active site on the enzyme. The active site is the specific region of the enzyme which combines with the substrate. The binding of the substrate to the enzyme causes changes in the distribution of electrons in the chemical bonds of the substrate and ultimately causes the reactions that lead to the formation of products. The products are released from the enzyme surface to regenerate the enzyme for another reaction cycle. The active site has a unique geometric shape that is complementary to the geometric shape of a substrate molecule, similar to the fit of puzzle pieces. This means that enzymes specifically react with only one or a very few similar compounds. Lock and Key Theory: The specific action of an enzyme with a single substrate can be explained using a Lock and Key analogy first postulated in 1894 by Emil Fischer. In this analogy, the lock is the enzyme and the key is the substrate. Only the correctly sized key (substrate) fits into the key hole (active site) of the lock (enzyme). Smaller keys, larger keys, or incorrectly positioned teeth on keys (incorrectly shaped or sized substrate molecules) do not fit into the lock (enzyme). Only the correctly shaped key opens a particular lock. This is illustrated in graphic on the left.

QUES: Using a diagram and in your own words, describe the various lock and key theory of enzyme

action in relation to a correct and incorrect substrate. Induced Fit Theory: Not all experimental evidence can be adequately explained by using the so-called rigid enzyme model assumed by the lock and key theory. For this reason, a modification called the induced-fit theory has been proposed. The induced-fit theory assumes that the substrate plays a role in determining the final shape of the enzyme and that the enzyme is partially flexible. This explains why certain compounds can bind to the enzyme but do not react because the enzyme has been distorted too much. Other molecules may be too small to induce

the proper alignment and therefore cannot react. Only the proper substrate is capable of inducing the proper alignment of the active site. In the graphic on the left, the substrate is represented by the magenta molecule, the enzyme protein is represented by the green and cyan colors. The cyan colored protein is used to more sharply define the active site. The protein chains are flexible and fit around the substrate.

Nature of Active Site and Substrate Interaction:

Enzymes have varying degrees of specificity. Some enzymes have absolute specificity for one substrate and no others, while other enzymes react with substrates with similar functional groups, side chains, or positions on a chain. The

least specific enzymes catalyze a reaction at a particular chemical bond regardless of other structural features. Much experimental work is devoted to gaining an understanding of the nature of the active site in an enzyme. Since enzymes are proteins, the nature of amino acid side chains in the vicinity of the active site is important. The specific amino acid side chains have been determined for many enzymes. The active site for carboxypeptidase A will be used to illustrate the principles involved as shown in the graphic on the left. The substrate (space filling gray,blue red) can interact with the active site through opposite charges, hydrogen bonding (shown in yellow), hydrophobic non-polar interaction, and coordinate covalent bonding to the metal ion activator as shown in magenta. The numbers behind the amino acids indicate the sequence position of the amino acid in the protein. The white lines represent the wire frames of the other amino acids in the enzyme. The carbonyl bond is activated by interaction with the Zn ions. This leads to the addition of -OH from water to the carbonyl to produce an acid and the ultimate rupture of the C-N bond.

Role of Enzymes in Biochemical Reactions Introduction - Enzyme Characteristics: A living system controls its activity through enzymes. An enzyme is a protein molecule that is a biological catalyst with three characteristics. First, the basic function of an enzyme is to increase the rate of a reaction. Most cellular reactions occur about a million times faster than they would in the absence of an enzyme. Second, most enzymes act specifically with only one reactant (called a substrate) to produce products. The third and most remarkable characteristic is that enzymes are regulated from a state of low activity to high activity and vice versa. Gradually, you will appreciate that the individuality of a living cell is due in large part to the unique set of some 3,000 enzymes that it is genetically programmed to produce. If even one enzyme is missing or defective, the results can be disastrous.

Much of the information about enzymes has been made possible because they can

be isolated from cells and made to work in a test tube environment. Extensive work has also been done with X-Ray diffraction techniques to elucidate the threedimensional structure of some enzymes. The ribbon and backbone form of carboxypeptidase is shown on the left. The substrate is shown in magenta.

Enzyme Parts List: The activity of an enzyme depends, at the minimum, on a specific protein chain. In many cases, the enzyme consists of the protein and a combination of one or more

parts called cofactors. This enzyme complex is usually simply referred to simply as the enzyme. Apoenzyme: The polypeptide or protein part of the enzyme is called the apoenzyme and may be inactive in its original synthesized structure. The inactive form of the apoenzyme is known as a proenzyme or zymogen. The proenzyme may contain several extra amino acids in the protein which are removed, and allows the final specific tertiary structure to be formed before it is activated as an apoenzyme. Cofactors: A cofactor is a non-protein substance which may be organic, and called a coenzyme. The coenzyme is often derived from a vitamin with specific examples discussed later. Another type of cofactor is an inorganic metal ion called a metal ion activator. The inorganic metal ions may be bonded through coordinate covalent bonds. The major reason for the nutritional requirement for minerals is to supply such metal ions as Zn+2, Mg+2, Mn+2, Fe+2, Cu+2, K+1, and Na+1 for use in enzymes as cofactors. Final Enzyme: The type of association between the cofactor and the apoenzymes varies. In some cases, the bonds are rather loose and both come together only during the course of a reaction. In other cases, they are firmly bound together by covalent bonds. The activating role of a cofactor is to either: activate the protein by changing its geometric shape, or by actually participating in the overall reaction. The overall enzyme contains a specific geometric shape called the active site where the reaction takes place. The molecule acted upon is called the substrate.

QUES: Using a diagram and in your own words, describe the various parts of the enzyme, i.e. What is an enzyme?

Enzyme Nomenclature and Classification: Enzymes are commonly named by adding a suffix "-ase" to the root name of the substrate molecule it is acting upon. For example, Lipase catalyzes the hydrolysis of a lipid triglyceride. Sucrase catalyzes the hydrolysis of sucrose into glucose and fructose. A few enzymes discovered before this naming system was devised are known by common names. Examples are pepsin, trypsin, and chymotrypsin which catalyzes the hydrolysis of proteins.

The latest systematic nomenclature system known as the International Enzyme Commission (IEC) system is based upon the type of reaction catalyzed. There are six broad groups of enzymes in this system as shown in table on the left. For example, when using this system, "urease" becomes "urea amidohydrolase." Do not be overly concerned about enzyme names, but be able to recognize a substance as an enzyme by its "-ase" ending. Some types of reactions which are being catalyzed will be self evident. Proteins - Introduction Introduction: Proteins are probably the most important class of biochemical molecules, although of course lipids and carbohydrates are also essential for life. Proteins are the basis for the major structural components of animal and human tissue. Proteins are natural polymer molecules consisting of amino acid units. The number of amino acids in proteins may range from two to several thousand. See Amino Acids. Primary Protein Structure: The primary protein structure is defined as the specific sequence of amino acids in the protein. In order to function properly, peptides and proteins must have the correct sequence of amino acids. In the section on peptide bonds, it was shown that a dipeptide consisting of two different amino acids could have two different sequences as in the example gly - ala or ala - gly. Remember that as written left to right in gly-ala, the glycine has the "free" amine

terminal end and alanine has the "free" carboxyl acid terminal end. If three different amino acids (gly, ala, leu) are used to make a tripeptide, how many different sequences are possible? There are six possible sequences: gly - ala - leu; gly - leu - ala; ala - gly - leu; ala - leu - gly; leu - ala - gly; leu - gly - ala. Review example: gly-ala-leu - Chime in new window In the protein hormone insulin, 51 amino acids are found. Using 51 amino acids there are 1.55 x 1066 different possible sequences. Many other proteins contain many more amino acids then insulin, but only the correct precise sequence is produced by the body. The procedure used to synthesize the correct sequence of amino acids in proteins is guided by the genetics of DNA and RNA.