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Enzymes Prof. Dr. Hedef Dhafir El-Yassin 2014 Enzymes Chaecteristics and Regulations Objectives: 1. To define enzymes 2. To state the major functions and characteristics of enzymes 3. To describe enzyme specificity and selectivity 4. To describe enzyme-substrate interaction 5. To list the major factors that regulate enzyme activity and describe how. Definition: An enzyme is a protein that catalyzes, or speeds up, a chemical reaction. Enzyme activity can be affected by other molecules: a. Inhibitors are naturally occurring or synthetic molecules that decrease or abolish enzyme activity; b. activators are molecules that increase activity. Certain RNAs also have catalytic activity, but to differentiate them from protein enzymes, they are referred to as RNA enzymes or ribozymes. Major functions and charecteristcs Enzymes are essential to life because most chemical reactions in biological cells would occur too slowly, or would lead to different products without enzymes. A malfunction (mutation, overproduction, underproduction or deletion) of a single enzyme can lead to a severe disease. For example, the most common type of phenylketonuria is caused by a single amino acid mutation in the enzyme phenylalanine hydroxylase, which catalyzes the first step in the degradation of phenylalanine. The resulting build-up of phenylalanine and related products can lead to mental retardation if the disease is untreated. ٢ Professor Dr.H.D.El-Yassin Ph.D. Post Doctorate 2014 Characteristics: • Like all catalysts, enzymes work by providing an alternate pathway of lower activation energy of a reaction, thus allowing the reaction to proceed much faster. Enzymes may speed up reactions by a million times. • Like any catalyst, an enzyme remains unaltered by the completed reaction and can therefore continue to function. • Because enzymes do not affect the relative energy between the products and reagents, they do not affect equilibrium of a reaction. • The advantage of enzymes compared to most other catalysts is their sterio-, regioand chemoselectivity and specificity. 3D Structure: In enzymes, as with other proteins, function is determined by structure. An enzyme can be: • A monomeric protein, i.e., containing only one polypeptide chain, typically one hundred or more amino acids; or • An oligomeric protein consisting of several polypeptide chains, different or identical, that acts together as a unit. ٣ Professor Dr.H.D.El-Yassin Ph.D. Post Doctorate 2014 As with any protein, each monomer is actually produced as a long, linear chain of amino acids, which folds in a particular fashion to produce a three-dimensional product. Individual monomers may then combine via non-covalent interactions to form a multimeric protein. Many enzymes can be unfolded or inactivated by heating, which destroys the threedimensional structure of the protein. Active site: Most enzymes are larger than the substrates they act on and only a very small portion of the enzyme come into direct contact with the substrate(s). This region is known as the active site of the enzyme. Some enzymes contain sites that bind cofactors, which are needed for catalysis. Certain enzymes have binding sites for small molecules, which are often direct or indirect products or substrates of the reaction catalyzed. This binding can serve to increase or decrease the enzyme's activity (depending on the molecule and enzyme), providing a means for feedback regulation. Specificity: Enzymes are usually specific to the reactions they catalyze and the substrates that are involved in these reactions. Factors responsible for this specificity are: 1. Shape, 2. charge complementarity's, and 3. Hydrophilic/hydrophobic character of enzyme and substrate. ٤ Professor Dr.H.D.El-Yassin Ph.D. Post Doctorate 2014 Mode of Enzyme Action There are two different views to explain the mode of enzyme action the lock and key hypothesis and the induced-fit hypothesis. 1. Lock and Key Hypothesis It was suggested by Emil Fischer in 1894. According to this view, the enzyme molecule operates by chemically uniting with the substrate molecule, forming an enzyme-substrate complex. The enzyme molecule provides a uniquely structured template on which the substrate molecules can become attached and interact subsequently. This brings about an interaction between the specific active sites in the enzyme molecule and the reactive sites in the substrate molecule. The enzyme now breaks down the substrate into products. The products initially remain attached to the enzyme for a short while forming an enzyme product complex. The products get released from the enzyme molecule subsequently. The enzyme is now ready to receive another substrate molecule again. Thus, the same enzyme can be used again and again. Example: Lock and Key Hypothesis ٥ Professor Dr.H.D.El-Yassin Ph.D. Post Doctorate 2014 2. Induced Fit Hypothesis It was proposed by Daniel Koshland in 1959. According to this hypothesis, there is an intermediate condition called transition state between the substrate and the products. It is highly unstable. When the substrate molecules bind to the enzyme molecule, a change is brought about in the active site to precisely fit the transition state (induced fit). This induced fit hold the substrates at the correct angle for the reaction to take place. The fact that an active site may also have a conformation to fit the product helps in explaining the role of enzymes in catalysing reversible reactions. Induced Fit Hypothesis Isoenzymes There are some enzymes which have slightly different molecular structure but exert similar catalytic action. Such enzymes are called isoenzymes or isozymes. More than 100 isozymes have been identified. The enzyme lactic dehydrogenase (LDH) in human skeletal muscle has five isozymes. Another kind of posttranslational modification is the cleavage and splicing of the polypeptide chain. Chymotrypsin, a digestive protease, is produced in inactive form as chymotrypsinogen in the pancreas and transported in this form to the stomach where it is activated. This prevents the enzyme from harmful digestion of the pancreas or other tissue. This type of inactive precursor to an enzyme is known as a zymogen. ٦ Professor Dr.H.D.El-Yassin Ph.D. Post Doctorate 2014 REGULATION OF ENZYMES FIRST. pH. A change in pH can alter the rates of enzyme-catalyzed reactions, with many enzymes exhibiting a bell-shaped curve when enzyme activity is plotted against pH (see Figure). Examples: Changes in pH can alter the following: a. The ionization state of the substrate or the enzyme-binding site for substrate b. The ionization state at the catalytic site on the enzyme c. Protein molecules so that their conformation and catalytic activity change ٧ Professor Dr.H.D.El-Yassin Ph.D. Post Doctorate 2014 SECOND. Temperature. The rate of an enzyme-catalyzed reaction usually increases with increasing temperature up to an optimum point (see Figure below), and then it decreases because enzymes are thermolabile. THIRD. Product inhibition. If the product accumulates, it can inhibit some enzymes. This form of control limits the rate of formation of the product when the product is underused. FOURTH. Covalent modification 1. Phosphorylation a. Effect on enzyme activity. In certain enzymes, the addition of a phosphate group to a specific amino acid residue [usually serine (Ser), tyrosine (Tyr), or threonine (Thr)] by specific protein kinases dramatically enhances or depresses activity. b. This modification is reversible. The phosphorylated enzyme may be dephosphorylated by specific phosphatases. 2. Nucleotidylation a. Effect on enzyme activity. The activities of certain enzymes are regulated by the reversible addition of a nucleotide (e.g., adenosine) to a specific amino acid. b. This modification is reversible. For example, an adenylated enzyme may be deadenylated by a specific enzyme. 3. Proteolytic cleavage. Certain enzymes are synthesized as proenzymes, or zymogens, which are inactive forms of enzymes that become active only after being cleaved at a specific site in their polypeptide chain by specific proteases. ٨ Professor Dr.H.D.El-Yassin Ph.D. Post Doctorate 2014 a. Many digestive enzymes that hydrolyze proteins (e.g., trypsin, pepsin) are synthesized as zymogens in the stomach and pancreas. b. Blood clotting is mediated by a series of proteolytic zymogen activities of several serum enzymes. FIFTH. Allosteric regulation of metabolic pathways. . A. Key enzymes that catalyze rate-limiting steps of metabolic pathways or that are responsible for major cellular processes must be regulated to maintain homeostasis of individual cells and the organism overall. B. Allosteric regulation refers to binding of a molecule to a site on the enzyme other than the active site and induces a subsequent change in shape of the enzyme causing an increase or decrease in its activity. C. Many allosteric enzymes have multiple subunits whose interaction accounts for their unusual kinetic properties. i. Enzymes that are subject to allosteric regulation by either positive or negative effectors exhibit cooperativity. ii. In the presence of positive cooperativity, a plot of v versus [S] shows sigmoidal kinetics, ie, is S-shaped (Figure). a. This kinetic behavior signifies that the enzyme's affinity for the substrate increases as a function of substrate loading. b. This is analogous to O2 binding by hemoglobin, in which O2 loading to one subunit facilitates O2 binding to the next subunit, and so on. Figure: Relationship between V; and [S] for a reaction catalyzed by an allosteric enzyme, showing the effects of positive and negative effectors. D. Feedback inhibition occurs when the end product of a metabolic pathway accumulates, binds to and inhibits a critical enzyme upstream in the pathway, either as a competitive inhibitor or an allosteric effector. ٩ Professor Dr.H.D.El-Yassin Ph.D. Post Doctorate 2014 Conclusions: 1. Enzymes are proteins that catalysis biological reactions. Some have slightly different molecular structure but exert similar catalytic action such enzymes are called isoenzymes. 2. Enzymes have specific characteristics and properties that make them similar to catalyst yet differ from them. 3. Enzymes are stereo specific and stereo selective 4. Several theories are discussed to explain the enzyme –substrate interactions among them are: a. Lock and key model b. Induced fit hypothesis 5. enzyme activities are regulated by several factors: 1 pH 2 Temperature 3 Product inhibition 4 Covalent modifications 5 Allosteric regulations of metabolic pathways ١٠ Professor Dr.H.D.El-Yassin Ph.D. Post Doctorate 2014