Enzymes-1 – Structure and Isoenzymes lecture NO : 1st MBBs Dr Muhammad Ramzan

Enzyme (E) – the definition A protein that catalyzes chemical reactions of other substances without itself being destroyed or altered upon the: Completion of the reactions. Enzymes lower the activation energy Enzymes are produced by the living organisms

Enzymes (E) – the background Intra/extra cellular All enzymes are proteins except a few catalytic RNAs- Ribozymes Defect or deficiency of an enzyme produces diseases called "inborn error of metabolism.“ like Lactose Intolerance The human body contains about 10,000 different enzymes Some enzymes work within the cell ( RNA polymerase) and some outside the cells like digestive enzymes The substance acted upon an by E is called a Substrate - S

Lactose intolerance

Biological significance of Enzymes Reduces activation energy At body temperature, very few biochemical reactions proceed at a significant rate without the presence of an E 1 Enzymes lower the activation energy of a catalyzed reaction 2 Thus increasing the rate of the reaction They do so in a step by step, highly efficient and safe manner 3 Enzymes uses activation energy which is the minimum energy required to start and complete a reaction

Enzyme lowers the activation energy

Properties of Enzymes Catalysis: act as protein catalysts - ↑ the velocity of reaction Activation energy: is reduced- energy to start and complete a reaction Active site binds and catalyze the S via side chains of its AAs Catalytic efficiency: E are efficient - Turn over no: is usually 103 – 108 Specificity: E is specific for specific S – Catalyzes 1 type of reaction Regulation: E activity is regulated by cell according to body needs Location of E- Compartmentalization – intra/extracellular

Parts of Enzymes(E) - 2 parts body and active site Enzymes are macromolecular proteins that are commonly globular in shape and variable in size - Each Enzyme has a specific area to bind the substrate by non covalently/temporary Essentially enzymes have 2 parts: Body of enzyme and Active site

Structure of enzyme – 2 parts Body and active site

Structure of enzymes – the body 1 Variable in size Enzymes are the globular proteins and their AA content vary from 62 to 2500 AA residues ( Fatty acid Synthase. FAS ) E are larger than S and some are grouped together to form enzyme complex like FAS/LDH Enzymes are long linear chains of AAs but their activities are due to their 3D Tertiary structures Apozyme is the protein portion (inactive E) while Halozyme is a protein with a cofactor/coenzyme (Active E)

Structure of enzyme Apozyme and Halonzyme

Structure of enzymes – the body

Structure of E – the active site 2 produced by protein folding Active sites are the hollows/cracks/ pockets on the surface of E and are produced by protein folding- 3D structure Active site contains 2-4 AA that are involved directly in S binding and catalysis (+ electrical+ chemical properties of AA side chains) This binding is temporary by non covalent bonds and is relieved after the completion of the catalysis S of just right shape and attractive groups/sequence of AAs can fit into these active sites The usual analogy for this is a key fitting exactly into the lock

Active site – the chemistry

Enzymes - Principle of working 1 E identifies and binds S Enzyme identifies the Substrate (S) and works by binding the Substrates so that they(S) are held in a : Particular Geometric configuration Forms Enzyme Substrate Complex (ESC) by binding E+S The binding is temporary (non covalent), E lowers the activation energy which is essential for the reaction to occur and complete

Enzymes – Principal of working 2 E controls the direction of reaction Both, the enzyme and products are released upon completion of the reaction and enzyme is available for Reuse Like other catalysts, an enzyme does not control the direction of the reaction. It is controlled by the cell itself Cell ↑the rates of forward and reverse reactions proportionally depending upon the body needs (Regulation)

Working of enzymes – the principals

Enzymes – Quantity and location Within and outside the cell Most enzymes are produced in tiny quantities and catalyze reactions that take place within the cells like . DNA/ RNA Polymerase and HMG CoA Synthase/Catalase Digestive enzymes, however, are produced in relatively large quantities and act outside the cells in the lumen of the GIT They cleave the dietary macromolecules like TG,Glycogen and Nucleic acids into smaller ones and monomers

Allosteric or Additional sites Allosteric enzymes Enzymes can also have sites other than active site that bind cofactors, which are needed for catalysis.(multi units) Some E also have binding sites for small molecules, which are often direct/ indirect products /S of the reaction catalyzed. This binding can serve to ↑or↓ (regulate) the enzyme's activity, providing a means for feedback regulation. They are the Allosteric sites and enzyme as Allosteric enzymes Glycogen Phosphorylase- that breaks down the glycogen when glucose is needed

Allosteric regulation

Allosteric sites and enzymes

Denaturation of enzymes – heat and pH Most enzymes can be denatured - unfolded and inactivated by heating or chemical denaturants / change of pH It disrupts the 3 structure of the protein (E) and results in the loss of function Denaturation of E may be reversible or irreversible depending upon the damage to the enzyme

E. Denaturation – high temperature

E denaturation- change of pH Reversible Denaturation

Isoenzymes/ Isozymes – the Definition difference in sequence of AAs Isoenzymes are generally the Multiple forms of the enzymes that differ in AA sequence but catalyze the : Same chemical reaction. These include Lactic Dehyrogenase (LDH) ,Creatine Kinase (CK) and Alkaline Phosphatase

Isoenzymes - Characteristics These display different electrophoretic mobility, regulatory and immunological properties Isozymes are the result of gene duplication These genes express the polypeptides chains that produce different subunits of the Isoenzymes Like CK has 2 Polypeptide subunits – M and B These include Lactic Dehydrogenase (LDH) ,Creatine Kinase (CK) and Alkaline Phosphatase

Isoenzymes – the clinical significance Different organs of the body contain the characteristic proportions of the different Isoenzymes The pattern of Isozymes in the plasma may identify the site of tissue damage like : Creatine Kinase has 3 forms and is present in various tissues The damage to these tissues releases  amount of respective CK Isoenzymes in circulation (Heart, brain and muscles)

Isoenzymes of CK (3) – the associated diseases There are 3 Isoenzymes of the CK including: CKMM In skeletal muscle diseases. CKMB cardiovascular diseases (MI) CKBB in brain and intestinal diseases like infarction

Creatine Kinase – Clinical importance Level of Creatine Kinase CK (CKMB) is commonly determined for the diagnosis of Myocardial infarction – MI Isoenzymes are particularly useful when ECG is difficult to interpret especially when there have been : Previous episodes of MI Same is true for Cardiac Troponin T and I