1. 1 ENZYMES ARE: Proteins (note that recent developments indicate that both RNA and antibodies may have catalytic activity, these are called ribozymes, and catalytic antibodies or abzymes, respectively) Biological catalysts, critical components of cell metabolism & biological processes Very efficient catalysts Like other catalysts, enzymes do not alter the position of equilibrium between substrates and products. However, unlike normal chemical reactions, enzymes are saturable. This means as more substrate is added, the reaction rate will increase, because more active sites become occupied.

2. 2 Very specific catalysts Reduce  G‡ for reaction (by binding the transition state) Subject to regulatory control of various sorts Carry out catalysis in a special region of the molecule, the active-site Exhibit special kinetics « All enzymes are proteins, with the exception of some small catalytic RNAs and RNA/protein complexes. « MW's range from 104 to 106 daltons. « May be single polypeptide chains, or oligomers of several subunits (most commonly oligomers are dimers, or tetramers, some multienzyme complexes as many as 48 protomers). « May have more than one activity associated with the same protein (i. e. there are some large enzymes which catalyze more than one reaction (frequently successive steps in a metabolic pathway). « Often contain a prosthetic group (or cofactor): Typical examples are: metal ions, heme, Fe-S clusters, coenzymes (e.g. NADH, FAD, FMN, PLP). « Coenzymes usually are vitamins, or derived from vitamins, and act as carriers (e.g. of H, e-, CO 2 ). « Enzymes are usually named after their substrate by adding ase, e. g. protease (proteinase), esterase,  -glucosidase, alcohol dehydrogenase,  -lactamase.

3. 3 Biological catalysts: Catalysts speed the rate of attainment of equilibrium by lowering the energy barrier between substrate and products. In other words a catalyst will increase the rate of a reaction but not affect the position of the reaction equilibrium. The catalyst is not used up in the reaction but is regenerated. Specificity: This is the second unique feature of enzymes as catalysts; they are very specific. A given enzyme will only catalyze one type of reaction for one type of compound, in some cases for only one compound. They are also very stereospecific, and produce no by-products. Free Energy of Activation Enzymes act as catalysts because they lower the free energy of activation (  G‡) for the reaction. They do this by a combination of raising the ground state  G of the substrate and lowering the  G of the transition state (TS) for the reaction, thereby decreasing the barrier for reaction to occur. The presence of the enzyme leads to a new (different) reaction pathway than for the uncatalyzed reaction. As we will see, the major way in which enzymes bring about their great rate enhancements is by tight binding of the TS. The height of the energy barrier is the free energy of activation.

4. 4 Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme kinetics and enzyme inhibition. Amounts of enzymes can either be expressed as molar amounts, as with any other chemical, or measured in terms of activity, in enzyme units. Enzyme activity = moles of substrate converted per unit time = rate × reaction volume. Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on conditions, which should be specified. The SI unit is the katal, 1 katal = 1 mol s-1, but this is an excessively large unit. A more practical and commonly-used value is 1 enzyme unit (EU) = 1 µmol min-1 (µ = micro, x 10-6). 1 U corresponds to 16.67 nanokatals. The specific activity of an enzyme is another common unit. This is the activity of an enzyme per milligram of total protein (expressed in µmol min- 1mg-1). Specific activity gives a measurement of the purity of the enzyme. Enzyme Assays All enzyme assays measure either the consumption of substrate or production of product over time. Methode: Spectrophotometric assays (colorimetric assays): the course of the reaction by measuring a change in how much light the assay solution absorbs. If this light is in the visible region you can actually see a change in the color of the assay, these are called Diagram of a single-beam UV/vis spectrophotometer

5. 5 Fluorimetric assays Fluorescence is when a molecule emits light of one wavelength after absorbing light of a different wavelength. Fluorometric assays use a difference in the fluorescence of substrate from product to measure the enzyme reaction. These assays are in general much more sensitive than spectrophotometric assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light. An example of these assays is again the use of the nucleotide coenzymes NADH and NADPH. The reduced forms are fluorescent and the oxidised forms non-fluorescent. Oxidation reactions can therefore be followed by a decrease in fluorescence and reduction reactions by an increase. Synthetic substrates that release a fluorescent dye in an enzyme- catalyzed reaction are also available, such as 4-methylumbelliferyl-β- D-glucuronide for assaying β-galactosidase. Factors to control in assays Salt Concentration: Most enzymes can not tolerate extremely high salt concentrations. The ions interfere with the weak ionic bonds of proteins. Typical enzymes are active in salt concentrations of 1-500 mM. Effects of Temperature: All enzymes work within a range of temperature specific to the organism. Increases in temperature generally lead to increases in reaction rates. There is a limit to the increase because higher temperatures lead to a sharp decrease in reaction rates. This is due to the denaturating (alteration) of protein structure resulting from the breakdown of the weak ionic and hydrogen bonding that stabilize the three dimensional structure of the enzyme. However, the idea of an "optimum" rate of an enzyme reaction is misleading, as the rate observed at any temperature is the product of two rates, the reaction rate and the denaturation rate.

6. 6 Effects of pH: Most enzymes are sensitive to pH and have specific ranges of activity. All have an optimum pH. The pH can stop enzyme activity by denaturating (altering) the three dimensional shape of the enzyme by breaking ionic, and hydrogen bonds. Substrate Saturation: Increasing the substrate concentration increases the rate of reaction (enzyme activity). However, enzyme saturation limits reaction rates. An enzyme is saturated when the active sites of all the molecules are occupied most of the time. At the saturation point, the reaction will not speed up, no matter how much additional substrate is added. The graph of the reaction rate will plateau. Amylase Amylase is the name given to glycoside hydrolase enzymes that break down starch into maltose molecules. They all act on α-1,4- glycosidic bonds. Starch is a mixture of amylose and amylopectin (usually in 20:80 or 30:70 ratios). These are both complex carbohydrate polymers of glucose Amylose structure Amylopectin structure

7. 7 Classification α -Amylase (EC 3.2.1.1) alternate names: 1,4- α -D-glucan glucanohydrolase; glycogenase). The α -amylases are calcium metalloenzymes, completely unable to function in the absence of calcium. By acting at random locations along the starch chain, α - amylase breaks down long-chain carbohydrates, ultimately yielding maltotriose and maltose from amylose, or maltose, glucose and "limit dextrin" from amylopectin. Because it can act anywhere on the substrate, α -amylase tends to be faster-acting than β -amylase. β -Amylase (EC 3.2.1.2) alternate names: 1,4- α -D-glucan maltohydrolase; glycogenase; saccharogen amylase. Another form of amylase, β -amylase is also synthesized by bacteria, fungi, and plants. Working from the non-reducing end, β -amylase catalyzes the hydrolysis of the second α -1,4 glycosidic bond, cleaving off two glucose units (maltose) at a time. is key to the production of malt. Many microbes also produce amylase to degrade extracellular starches. γ-Amylase (EC 3.2.1.3 ) alternative names: Glucan 1,4-α-glucosidase; amyloglucosidase; Exo- 1,4-α-glucosidase; glucoamylase; lysosomal α-glucosidase; 1,4-α-D- glucan glucohydrolase) In addition to cleaving the last α(1-4)glycosidic linkages at the nonreducing end of amylose and amylopectin, yielding glucose, γ-amylase will cleave α(1-6) glycosidic linkages. Pullulanase (EC 3.2.1.41) is also known as pullulan-6-glucanohydrolase (Debranching enzyme). Its substrate, pullulan, is regarded as a chain of maltotriose units linked by alpha-1,6-glycosidic bonds. Pullulanase will hydrolytically cleave pullulan (alpha-glucan polysaccharides). Pullulanase is a specific kind of, an amylolytic exoenzyme, that degrades pullulan a polysaccharide polymerconsisting of maltotriose units, also known as α - 1,4- ; α -1,6-glucan. It is produced as an extracellular, cell surface-anchored lipoprotein by Gram- negative bacteria of the genus Klebsiella. Type I pullulanases specifically attack α-1,6 linkages, while type II pullulanases are also able to hydrolyse α- 1,4 linkages. It is also produced by some other bacteria and archaea. Pullulanase is used as a detergent in biotechnology.