MBC 302: BASIC ENZYMOLOGY & IMMUNOCHEMISTRY 3 UNITS; C Mr Kweki Classification and nomenclature of enzymes. Mechanisms of enzymes-catalyzed reactions. Effects of temperature, (pH), ions and inhibitors on enzymes. Active sites of enzymes. Concepts of ‘cooperativity. Estimation of kinetic parameters — enzymes activities, Km, Vmax, Ki, et. Miss Vanessa Isoenzymes, zymogen activation, digestive enzymes. Production, isolation, purification and characterization of enzymes from animal and plant tissues. Diagnostic enzymes. Prof Mordi Concept and types of immunity. Immunogens, antigens and haptens. Biochemical requirements. for immunogenicity. Antibodies; class, structure, synthesis and function. Antigen-antibody reactions. The biochemistry of hypersensitivity and allergy. Major histocompatibility complex and immunological tolerance Dr Orororo Autoimmunity and concept of autoantibodies. Biochemistry of the complement system. Applications of immune-biochemisty in clinical and biological studies; production of specific antibody. Radio-immuno assay (RIA) and blood group serology. An outline of Immuno- diffusion techniques.UNIT ONE: NATURE OF ENZYMES WHAT ARE ENZYMES Enzymes are biological catalysts. They increase the rate of chemical reactions taking place within living cells without themselves suffering any overall change. The reactants of enzyme-catalyzed reactions are termed substrates. Each enzyme is quite specific in character, acting on a particular substrate or substrates to produce a particular product or products. All enzymes are proteins. However, without the presence of a non-protein component called a cofactor, many enzyme proteins lack catalytic activity. When this isthe case, the inactive protein component of an enzzyme is termed the apoenzyme, and the active enzyme, including cofactor, the holoenzyme. The cofactor may be anorganic molecule, when it is known as a coenzyme, or it may be a metal ion. Some enzymes bind cofactors more tightly than others. When a cofactor is bound so tightly that it is difficult to remove without damaging the enzyme, itis sometimes called a prosthetic group. To summarize diagrammatically oo Simple Enzyme oe ~ Apoenzyme Holo Enzyme a aN. Cofactor A BRIEF HISTORY OF ENZYMES. Unvil the nineteenth century, it was considered that processes such as the souring of mill and the fermentation of sugar to alcohol could only take place through the action of a livitlg organism. in 1833, the active agent breaking down the sugar was partially isolated and given the name diastase (now known as amylase). A litte later, a substance which digested dietary protein was extracted from gastric juice and called pepsin. These and other active preparations were given the generaltame ferments, Justus von Liebig recognized that these ferments could be non-living materials obtained from living cells, but Louis Pasteur and others still maintained that ferments must contain living material, |While this dispute continued, the term ferment was gradually replaced by the name enzyme. This \was first proposedby Wilhelm Kfthne in 1878, and comes from the Greek, enzume meaning ‘in ‘yeas’, Appropriately, it was in yeast that a factor was discovered which settled the argument in favour of the inanimate theory of catalysis: brothers Eduard and Hans Buchner showed, in 1897, that sugar fermentation could take place when a yeast cell extract was added even though no living cells were present. In 1926, James Sumner erystallized urease from jack-bean extracts and, in the next few years, many other enzymes were purified and crystallized. Once pure enzymes were available, their structure and properties could be determined, and the findings form the material for most ofthis book. Today, enzymes still form a major subject for academic research. They are investigated in hospitals as an aid to diagnosis and, because of their specificity of action, are of great value as analytical reagents. Enzymes are still widely used in industry, continuing and ‘extending many processes which have been used since the dawn of history. ‘THE NAMING OF ENZYMES ‘There is long tradition of giving enzymes names ending in '-ase’. The only major exceptions to this are the proteolytic enzymes, ie. ones involved in the breakdown of proteins, whose names usually end with '-in', e.. trypsin. The names of enzymes usually indicate the substrate involved. Thus, lactase catalyses the hydrolysis of the disaccharide lactose to its component ‘monosaccharides, glucose and galactose: Ci HnOn +20. <———* Cai20s + CHO ‘The name lactase is a contraction of the clumsy, but more precise, lactosase. The former is used! because it sounds better but it introduces a possible trap for the unwary because it could exsily suggest an enzyme acting on the substrate lactate. There is nothing in the name of this enzyme oF 2many others to indicate the type of reaction being catalysed. Fumarase, for example, by analogy with lactase might be supposed to catalyse a hydrolytic reaction, but, in fact, it hydrates fumarate to form malate: ‘The names of other enzymes, e.g. transearboxylase, indicate the nature of the reaction without specifying the substrates (which in the case of transcarboxylase are methylmalonyl-CoA and pyruvate). Some names, such as eatalase, indicate neither the substrate nor the reaction (catalase ‘mediates the decomposition of hydrogen peroxide). Needless to say, whenever a new enzyme has been characterized, great care has usually been taken not to give it exactly the same name as an enzyme catalyzing a different reaction. Also, the names of many enzymes make clear the substrate and the nature of the reaction being catalyzed. For example, theres little ambiguity about the reaction catalyzed by malate dehydrogenase. This ‘enzyme mediates the removal of hydrogen from malate to produce oxaloacetate: However, malate dehydrogenase, like many other enzymes, has been known by more than one rname. So, because ofthe lack of consistency inthe nomenclature, it became apparent as the list of known enzymes rapidly grew that there was a need for a systematic way of naming and classifying enzymes. A commission was appointed by the International Union of Biochemistry (later re-named the Intemational Union of Biochemistry and Molecular Biology, IUBMB), and its report ‘Published in 1964, forms the basis of the currently accepted system. Revised editions of the report ‘were published in 1972, 1978, 1984 and 1992. An electronic version is now maintained by the IUBMB on an accessible web-site, and this is updated on a regular bass. ‘The Enzyme Commission's system of classification ‘The Enzyme Commission divided enzymes into six main classes, on the basis of the total reaction catalysed. The classification of enzyme was described in 1961 by enzyme commission of the Intemational Union of Biochemistry (TUB). According to this classification, each enzyme is characterized by « code number called enzyme code number or EC’ number, consisting of four digits. According to the TUB system, enzymes are classified into six major classes as followsClassification And Examples of Enzymes With Their Main Class sino. | Enzyme | Class of Enzymes Subclasses of | Examples code(EO) Enyzmes T [ECT | Oxidoreductases Dehydrogenases | Lactate dehydrogenase Reductases pH) Oxidases Glucose ‘6-phosphate Peroxidases dehydrogenase (G-6-PD) Cytochrome oxidase 2 [EC-2 | Transferases ‘Amino transferase | Aspartate | ortransaminase | aminotransaminase(AST) Kinase Alanine aminotransaminase Transcarboxylase | (ALT) Hexokinase 3 [ECS | Hydrolases ‘Acid phosphatase | Lipase All digestive | a-Amylase enzymes like | Trypsin amylase, pepsin, | Lactase typsin, Sucrase | chymotrypsin | Pepsin |6 [EC | Ligases. ‘Thiokinase Glutamine synthetase DNA ligase Pyruvate carboxylase Chelatases DNA ligases Synthetase Oxidoreductases ‘These enzymes catalyze oxidation-reduetion reactions which can be illustrated schematically as follows ‘ A +) Be———+Bi+ a (red) (0x) (red) (ox) Specific Example Nap" He Pyruvate ‘Transferases Enzymes that catalyze the transfer of amino, carboxyl, methyl or phosphoryl groups from one molecule to another are called transferases. These reactions can be illustrated as follows AY +B4+———ra + BY Notable exampleALT Alanine + a-Ketoglutarate === Pyruvate + Glutamate PLP where, ALT:: Alanine aminotransferase and PLP : Pyridoxal phosphate (coenzyme) Hydrolases Enzymes of this class catalyze the cleavage of C-O, C-N, C-C and some other bonds with the addition of water. These can be illustrated as follows X-Y+HO «______+X-OH + BH Notable Example Glucose-6-phosphate + Hxo ————__—>> Glucose + Pi Lyases Lyases cleavage C-O, C-C, and C-N bonds via other means than hydrolysis or oxidat nto produce compounds with double bonds or catalyze the reverse reaction, by the addition of « group to.a double bond. In cases where reverse reaction is important, then synthase, (not synthetase of group EC-6) i used inthe name. This type of reaction is illustrated as follows: atx ‘Wyases) fy Siig) 5 +X-YCommon exampl Fructose 1,6- <——> Glyceraldehyde- 3-phosphate « bisphosphate Dihydroxy actone phosphate Tsomerases Isomerases are groups of enzymes that catalyze intramolecular structural rearrangement in & molecule, They are called epimerases, isomerases, or mutases, depending on the (ype of isomerism catalyzed. This reaction can be illustrated as follows: ABC + CAB ‘Common example Ligases (Synthetases) Ligases are notable for the joining of two molecules coupled with the hydrolysis of ATP. The reaction is illustrated as follows: A+B+AIP > AB +ADP + Pi‘Common example Glutamate + NHy + ATP > Glutamine + ADP * Pi PROPERTIES OF ENZYME, 1) Active si Enzyme molecules have an active site, o cleft, in which amino acid chains form a 3-limensional surface that is complementary to the substrate, The active site binds to the substrate, and (he enzyme substrate (ES) complex is converted into an enzyme-produet (EP); which then dissociate into an enzyme and a product. Each enzyme has one ot more "active sites” where it can take the substrate. These "active sites" can include free hydroxyl groups of serine and tyrosine, as well asthe phenolic and sulfhydryl groups of SH-hiol and imidazole groups of cysteine and histidine to interact with substrates. It is also possible that the active site (Catalytic sie) is different from the binding site in which case they are situated closely together in the enzyme molec 2), Catalytic efficency/ Enzyme turnover number Enzyme-catalyzed reactions are the most efficent, taking between 103 and 108 times ‘unaccelerated reactions. Fach enzyme molecule is usually able to convert between 100 and 1000 substrate molecules into a product every second. The enzyme tum over number is the numbe substrate molecules converted in a given unit time (Carbonic Anhydrase being the fastest enzyme 3). Specificity of Enzyme Action. Specificity refers to the ability of an enzyme to discriminate between two competing sub Enzymes are highly specific both in the reaction catalyzed anSpecificity makes it possible for a number of enzymes to co-exist inthe cell without interfering in ‘each other's actions. Types of Specificity ‘The following types of specificity have been recognized: 1. Substrate specificity 2. Reaction specificity 3. Stereo specificity. 1), Substrate Specificity ‘There ae the following types of substrate specificity 4). Absolute substrate specificity Certain enzymes will act on only one substrate and catalyze one reaction, ¢. Glucokinase, lactase, ‘urease, ete. Glucose ————> Glucose-6-phosphate Lactose Glucose +Galactose Ura > Ammonia + co, »), Relative substrate specificity In this case, the enzyme acts on more than one substrate. The relative substrate specificity is of two types: a. Group specificity b. Bond specificity 1). Group specificity, the enzyme acts on more than one substrate containing a particular group, ce chymotrypsin acts on several proteins by hydrolyzing peptide bonds attached to arom acids. Trypsin hydrolyzes peptide linkages involving arginine or lysine ii), Bond specificity, here, the enzyme acts on more than one substrate containing « particulier kind of bond, e.g. salivary a-amylase cleaves a-{I-4) glycosidic bonds of earbolydra hydrolyzes ester bonds of lipidsBroad substrate specificity an enzyme acts on more than one structurally related substrates, e-8, hexokinase In this case, catalyzes the phosphorylation of more than one kind of hexoses such as glucose, fructose and mannose 2), Reaction Specificity Inthis case, an enzyme is speci ‘one type of reaction. For example, pyruvate can undergo several reactions: catalyzed by a separate enzyme, which catalyzes only that react and none other. fic to a particular reaction but not to substrate (8) and catalyzes only Each reaction is Fig 9. Example of reaction specificity 3), Stereo Specificity ‘Numbers of enzymes show specificity towards stereoisomers, ic. they act on only one type of isomer, For example, L-lactate dehydrogenase will act only on L-lactic acid and not D-lactic aid Likewise, L-amino acid oxidase and D-amino acid oxidase are distinct enzymes which act only on Land D-amino acids respectively. D-glucose oxidase can similarly act only on D- glucose and not ‘on L-glucose. Salivary a-amylase acts on the a-1,4 glycoside linkage and is inactive on b-I4 slycoside linkage. Isomerase and epimerase do not show stereospecificity 4, Regulation: Enzyme activity can be regulated that i, enzyme can be, activated or inhibited so thatthe rate of product formation responds to the needs of the cel 5). Zymogens: These are inactive form of enzyme. Some enzymes are produced in mature in am inactive form which can be activated when they are required. Such type of enzymes are called ‘Zymogens (Proenzymes).Many of the digestive enzymes and enzymes concemed with blood ‘coagulation are in this group. Examples: Pepsinogen - This zymogen is from gastric juice. Whem required Pepsinogen converts to Pepsin. Trypsinogen - This zymogen is found in the puncrewtie Juice, and when itis required gets converted to trypsin. The activation is brought about by specie nions or by other enzymes that are proteolytic, Zymogen forms of enzymes a protective mechanism to preven auto digestion ofssue producing the digestive enzymes and to prevent intravascular coagulation of blood. Isoenzymes (Isozymes Isoenzymes or isozymes are multiple forms isomers) of the same enzyme action. Isoenzymes show different chemical and physical that catalyze the same biochemical re properties like electrophoreiie mobility and kinetic properties. Not all enzymes have isoenzyins In fact, it was found that only those enzymes, which are active in polymeric form demonstrate isoenzyme. For example: 1. Lactate dehydrogenase (LDH) 2, Creatine kinase (CK) (formerly called creatine Alkaline phosphokinase (CPK). other examples of isoenzymes are Acid phosphatase, phosphatase, Amylase, Hexokinase ete Lactate Dehydrogenase (LDH) Lactate dehydrogenase is a tetrameric enzyme that catalyzes the oxidation of L-lactae to pyruvate It has five isoenzymes: -LDHI -LDH2 -LDH3 -LDHS -LDHS. LDH is a tetramer, made up of two types of polypeptide (muscle) type and H (heart) type. five combinations are possible with varying ratios of two kinds of poly LDH can be detected by electrophoresis as they have differen is the fastest moving fraction towards the anode, it pr Erythrocytes. However, LDHS is the least moving isoenz; and in skeletal muscle.