MSA University Biochemistry Lecture 6: Hemoglobin and Enzymes PDF

Biochemistry department Lecture 6: Hemoglobin and Enzymes Dr Samar Samir elkhateeb Lecturer Biochemistry and Genetics Faculty of Dentistry –MSA university [email protected] LECTURE OUTLINE Prophyrin structure. Heme proteins. Hemoglobin structure. Types of heamoglobin. Enzymes and t...

Biochemistry department Lecture 6: Hemoglobin and Enzymes Dr Samar Samir elkhateeb Lecturer Biochemistry and Genetics Faculty of Dentistry –MSA university [email protected] LECTURE OUTLINE Prophyrin structure. Heme proteins. Hemoglobin structure. Types of heamoglobin. Enzymes and their classification. Enzyme active site. Factors affecting the rate of enzyme. Regulation of Enzyme activity. Importance of enzymology. LECTURE ILOs By the end of the lecture the students will be able to : Know what is the prophyrins and their role. know the chemical composition of hemoglobin. know the chemical nature of Enzymes. know Models of Enzyme-Substrate binding. Know Factors affecting the rate of enzyme catalyzed reactions andRegulation of Enzyme activity. Know the difference between functional and non- functional plasma enzymes. Heamoglobin and prophyrin Porphyrin: Porphyrins are termed “pigments of life”. They play a wide range of functions in oxygen carriers, catalytic cycles and photosynthetic processes. Porphyrins are highly coloured cyclic tetrapyrrolic pigments formed by the linkage of four pyrrole rings through methene bridges (–HC=). Metalloporphyrins The porphyrins have a characteristic property of formation of complexes with metal ions bound to the nitrogen atom of the pyrrole rings. The porphyrins containing the metal atom are called metalloporphyrins. Various examples of metalloproteins occurring in nature are: Iron containing porphyrins: Heme proteins (hemoglobin, myoglobin, cytochrome, enzymes catalase and peroxidase). Magnesium containing porphyrin: Chlorophyll. Cobalt containing porphyrins: Vitamin B12.  Heme proteins: Hemoglobin Hb is a hemoprotein whose primary function is to transport oxygen from the lungs to the body tissue. Heme structure: Heme is formed by the complexation of Fe2+ ion with the porphyrin ring by ferrochelatase enzyme.  Globin Structure: 1. Primary structure: Hb consists of 4 polypeptide chains (each 2 chains are identical to each others). HbA (represents at least 96% of the total Hb) consists of 2 α- and 2 β-chains that are 141 and 146 amino acid residues in length, respectively. 2. Secondary Structure: Approximately 75% to 80% of the polypeptide chains of the α- and β- chains are arranged in helices. 3. Tertiary Structure: The heme group of hemoglobin sits in a hydrophobic pocket (formed of non polar amino acids) in the molecule to protect Fe2+ from oxidation. 4. Quaternary Structure: The quaternary structure of Hb results from the attachment of the four globin chains to each other.  Functions: Hemoglobin is found exclusively in red blood cells (RBCs), where its main function is to : 1. transport oxygen (O2) from the lungs to the capillaries of the tissues. 2. Hemoglobin transports H+ and CO2 from the tissues to the lungs.  Heme proteins: Hemoglobin Forms of Hemoglobin 1- Oxyhemoglobin: Hemoglobin can bind four oxygen molecules; one at each of its four heme groups. 2- Carbaminohemoglobin: Most of the CO2 produced in metabolism is hydrated and transported as bicarbonate ion. However, some CO2 is carried as carbamate bound to the Nterminal amino groups of hemoglobin. The binding of CO2 stabilizes the T-form of hemoglobin, resulting in a decrease in its affinity for oxygen. In the lungs, CO2 dissociates from the hemoglobin, and is released in the breath 3- Modified hemoglobins: a) Carboxyhemoglobin is: formed by the preferential attachment of carbon monoxide (CO) over oxygen (affinity of Hb for CO is 220 times greater than for oxygen to Hb). As a result, the affected Hb is unable to carry oxygen to the tissues. b) Methemoglobin The iron is oxidized to the ferric state (Fe3+ ) by toxic agents. Methemoglobin is not an oxygen carrier. c) Glycated haemoglobin (Hemoglobin A1c ) Under physiologic conditions, HbA is slowly and nonenzymically and irreversibly glycosylated, the extent of glycosylation being dependent on the plasma concentration of glucose. Glycated Hb is used in assessing average blood glucose levels in diabetic patients  4- Abnormal Hemoglobins (Hemoglobinopathies): a) Thalassemias: It occur as a result of mutations in globin genes causing unbalanced globin structure. It can involve α-chains (α-thalassemia) or β-chains (β- thalassemia). The consequences include ineffective erythropoiesis, hemolysis and anemia. b) Hemoglobin S: (Sickle cell anemia) It is caused by substitution of polar glutamate by non polar valine in 6 th position of β chains of Hb A. This results on formation of sticky hydrophobic patches on surface of β chains which leads to polymerization of Hb S in the form of insoluble fibers that distort the shape of RBC (sickling). Sickled cells are more fragile than normal RBCs, so they have shorter life span which leads to sickle cell anemia. Myoglobin (Mb) is a heme protein, found primarily in cardiac and red skeletal muscles. Mb function is storage of oxygen. Mb structure very closely resembles a single Hb subunit which allows the Mb to bind to only one molecule of oxygen. Enzymes Introduction Virtually all reactions occurring in the body are catalyzed by enzymes (catalytic proteins) that are synthesized within living cells. Similar to chemical catalysts, enzymes accelerate the rate of chemical reactions occurring in the biological system without being themselves changed in the overall process. Unlike chemical catalysts enzymes possess: a.Specificity: reaction-specific. b.Protein conformation: they allow the reactions in the biologic system to proceed rapidly under physiologic conditions of temperature & pH but they are denatured under extreme pH and high temperature (enzymes are proteins). c.Catalytic power: highly efficient proceeding from 103 to 1018 times faster than chemically catalyzed reactions. In any enzymatic reaction, the substances whose reaction is catalyzed by an enzyme are termed substrates. Classification of Enzymes Enzymes are classified into six classes according to reaction types: Class Type Of Reaction Examples Oxido-reductases Oxidation-reduction Dehydrogenases, Oxidases, Catalase Transferases Group tranfer Kinases Hydrolases Hydrolytic cleavage Proteases, Lipases Lyases Removal of groups To form Fumarase double bonds Isomerases Isomerization Mutases Ligases (Synthases) Synthetic reactions where Carboxyalses 2 molecules are joined Chemical Nature of Enzymes Simple proteins: when the native conformation of the protein is only required for activity e.g. urease, pepsin, trypsin. Conjugated protein:ctivity requires the presence of non protein component called cofactors may be 1. Metal ions or 2. Coenzymes:  Enzyme-catalyzed reaction All enzymes contain a special pocket or cleft called active site. The active site binds the substrate, forming ES complex, which then subsequently dissociates to a product and enzyme. E +S ➔ ES ➔ E + P E is the enzyme S is the substrate ES is the enzyme substrate complex P is the product Features of Enzyme active site The active site is formed only of few amino acid residues (~ 5 residues). It is not a point or a line but it is a three dimensional entity formed by groups that come from different parts of the linear amino acid sequence. Substrate is bound to enzyme by multiple weak attractions (non- covalent bonds). Active site is cleft from which water is usually excluded, this enhances the binding of substrate. Models of Enzyme-Substrate binding Lock and Key Model Induced-Fit Model (Fischer Model): (Koshland Model): Active site is rigid. Active site is flexible (not It is like a "lock and key" fully formed), however the in which the substrate as correct substrate can a key fits to the active recognize it. site (lock), just as a key When the substrate goes into proper lock. approaches the enzyme, a conformational change essential for binding & catalysis takes place. Factors affecting the rate of enzyme catalyzed reactions 1. Substrate concentration 2. Enzyme concentration 3. Temperature 4. pH 5. Time and product 6. Enzyme inhibitors  1- Substrate Concentration At low substrate conc. [S], substrate is the rate limiting factor and the reaction velocity (V0 ) is linearly proportional to [S]. As [S] gets large, the enzyme becomes the ratelimiting factor (all enzyme molecules are 'busy' operating on the substrate) and the rate of reaction is dependent on the amount of enzyme, but not dependent on the amount of substrate. At high [S], V0 is constant independent of [S] and equals Vmax. Maximum Velocity (Vmax): the rate of the reaction when all the enzymes are saturated. i.e. all the enzymes are bound to substrate. 2- Enzyme concentration The rate of the reaction (V) is directly proportional to the enzyme concentration (E). 3- Temperature Temperature Coefficient (Q10) is the increase in reaction rate with a 10°C rise in temperature. 4- pH Extreme pH levels will produce denaturation (active site is distorted and the substrate molecules will no longer fit in it) 5- Time and Product By time, the amount of substrate is reduced and the product is accumulated. The accumulated product, by law mass of action, increases the rate of reverse reaction. In the biological system however, the product is usually removed as it becomes a substrate for the following enzyme in a metabolic pathway. 6- Enzyme Inhibitors ✓Competitive Inhibitors : Both inhibitor and substrate are competing for the same site. Competitive inhibitor effect can be overcome by increasing [S]. ✓Non competitive Inhibitors:Inhibitor and substrate bind at different sites on the enzyme ✓Uncompetitive Inhibitors :They bind only to the ES complex without binding to the free enzyme. Regulation of Enzyme activity I. Control of enzyme level: a) Induction: It is the increase in the production of the enzyme. This increase is done by hormonal activation of its gene expression, e.g: insulin b) Repression and depression It is the decrease in the production of the enzyme. This decrease is done by hormonal deactivation of its gene expression, e.g insulin c) Proenzymes : They are enzymes which are synthesized in catalytically inactive form. e.g Digestive proteolytic enzymes (Pepsinogen➔ pepsin) They are converted into active form by splitting of small peptide chain covering the active site. Regulation of Enzyme activity cont. II. Modulation of the catalytic efficiency of the existing enzymes a) Covalent modification: Reversible interconversion between their active and inactive forms through covalent modification of enzyme structure The most common way of such type is the phosphorylation of enzymes. This can be done by covalent attachment of a phosphate group to a serine, threonine or tyrosine residues of the enzyme, e.g: protein kinases and phosphatases b) Non-covalent modification: t is done by non-covalent attachment of certain metabolite (effector) at an allosteric site.  Regulation of Enzyme activity cont. II. Modulation of the catalytic efficiency of the existing enzymes a) Covalent modification: Reversible interconversion between their active and inactive forms through covalent modification of enzyme structure The most common way of such type is the phosphorylation of enzymes. This can be done by covalent attachment of a phosphate group to a serine, threonine or tyrosine residues of the enzyme, e.g: protein kinases and phosphatases b) Non-covalent modification: t is done by non-covalent attachment of certain metabolite (effector) at an allosteric site.  Clinical Enzymology Enzymes are commonly employed in medicine in three aspects: 1. Analytical reagent for determination of various constituents of biological fluids. 2. As index of pathology or disease. 3. Therapeutic agent. Enzymes as a diagnostic agents for diseases There are two types of plasma enzymes: 1. Functional plasma enzymes: They are present all the time in the circulation of normal individuals. Their substrates are present in plasma either continuously or intermittently. These enzymes perform a certain physiological function e.g. proenzymes of blood clotting. These enzymes are present in blood in equivalent or higher concentrations than in tissues. Enzymes as a diagnostic agents for diseases 2. Non-functional plasma enzymes: They perform no physiological function in blood. Their substrates are absent from blood. Their level in blood is extremely low in normal individuals. Thus, their presence in plasma at levels higher than their normal values suggests tissue destruction.  References: Rayati S, Malekmohammadi S (2016). "Catalytic activity of multi-wall carbon nanotube supported manganese (III) porphyrin: an efficient, selective and reusable catalyst for oxidation of alkenes and alkanes with urea–hydrogen peroxide". Journal of Experimental Nanoscience. 11 (11): 872. Bibcode:2016JENan..11..872R. doi:10.1080/17458080.2016.1179802 Ivanov AS, Boldyrev AI (August 2014). "Deciphering aromaticity in porphyrinoids via adaptive natural density partitioning". Organic & Biomolecular Chemistry. 12 (32): 6145– 6150. doi:10.1039/C4OB01018C Walker, F. Ann; Simonis, Ursula (2011). "Iron Porphyrin Chemistry". Encyclopedia of Inorganic and Bioinorganic Chemistry. doi:10.1002/9781119951438.eibc0104 Patton, Kevin T. (2015-02-10). Anatomy and Physiology. Elsevier Health Sciences. ISBN 978- 0-323-31687-3. Archived from the original on 2016-04-26 Epstein, F. H.; Hsia, C. C. W. (1998). "Respiratory Function of Hemoglobin". New England Journal of Medicine. 338 (4): 239–47. doi:10.1056/NEJM199801223380407 aha, D.; Reddy, K. V. R.; Patgaonkar, M.; Ayyar, K.; Bashir, T.; Shroff, A. (2014). "Hemoglobin Expression in Nonerythroid Cells: Novel or Ubiquitous?". International Journal of Inflammation. 2014 (803237): 1–8. doi:10.1155/2014/803237