Enzymology PDF

Teresa N. Villanueva RMT, MACT branch of medicine which deals with the study of enzymes and their importance in the diagnosis and treatment of diseases CELLULAR CATALYSTS Biologic catalysts that cause reactions in the body to take place 1. Complicated type of protein in terms of bo...

Teresa N. Villanueva RMT, MACT branch of medicine which deals with the study of enzymes and their importance in the diagnosis and treatment of diseases CELLULAR CATALYSTS Biologic catalysts that cause reactions in the body to take place 1. Complicated type of protein in terms of both structure and function 2. Easily denatured with varying molecular weight & mass 3. Amphoteric 4. Operate at high rates 5. Reactions catalyzed are frequently REVERSIBLE 6. Usually operates with the assistance of a non-protein COFACTOR 7. Synthesized in an inactive state (ZYMOGEN/PROENZYME) 8. Changes in enzyme concentration reflect changes in state of health of a tissue 1)COENZYME – non-protein organic biochemical - Essential to the catalytic activity as a CO-SUBSTRATE - E.g. NAD, Pyridoxal phosphate 2) ACTIVATOR - Inorganic ionic cofactor - Metabolic regulator of enzyme reaction - E.g. Mg++, Ca++, K+, Zn++ 3) HOLOENZYME - the combined enzyme & coenzyme 4) APOENZYME - Enzyme without a cofactor 5) PROSTHETIC GROUP - A coenzyme that cannot be separated from an enzyme - E.g. Pyridoxal phosphate in transaminase reaction 6) METALLOENZYME - Inorganic activators existing as a part of the enzyme molecule 7) SUBSTRATE - Substance acted upon by an enzyme & is converted into a new substance 8) PRODUCT - transformed substrate FOUR PROTEIN STRUCTURES A. PRIMARY STRUCTURE - sequence of amino acids joined by peptide bonds to form a polypeptide chain B. SECONDARY STRUCTURE - Conformation of the segments of polypeptide chain - Alpha-helix ; beta-pleated sheet - Maintained by hydrogen bond C. TERTIARY STRUCTURE - Arises from the interactions among side chains/groups of the polypeptide chain - Bent and folded structure - Maintained by covalent disulfide bond The 2° and 3° structures: most important configurations of an enzyme responsible for the conformation of the active site ACTIVE SITE – area on the enzyme where the substrate attaches & undergoes transformation D. QUARTERNARY STRUCTURE - Separate bent & folded structures are put together to form a functional unit - Examples: Hemoglobin – consists of 4 polypeptide units Enzyme variants – LDH, Creatine kinase Based on catalytic activity of an enzyme The International Union of Biochemistry (IUB) Enzyme Commission categorized all enzymes into six (6) classes I. OXIDOREDUCTASES  Catalyze oxidation-reduction reactions  Older names: Dehydrogenases & Oxidases  Investigation of cardiac and liver disorders  Examples: Glucose oxidase Cytochrome oxidase Lactate dehydrogenase Isocitrate dehydrogenase II. TRANSFERASES - Move an intact group of atoms from one molecule to another (NH2 or PO4) - Lab diagnosis of liver & muscle damage - Examples: Aspartate aminotransferase Alanine aminotransferase Creatine kinase  ATP + Creatine ADP + Creatine PO4 III. HYDROLASES  Splits molecules in the presence of water  Three (3) Groups: A. Esterases: ACP, ALP, Lipase B. Peptidases: Leucine aminopeptidase, Pepsin C. Glycosidases: Amylase, Amylo-1,6- glucosidase IV. LYASES  Responsible for splitting molecules or breaking of bonds (C to C; C to O; C to N, etc.)  Assayed in skeletal muscle disorders  Examples: Aldolase Glutamate decarboxylase Pyruvate decarboxylase V. ISOMERASES  conversion of one isomer to another  Transformation from: Cis to Trans L to D forms Aldehyde to Ketone  All reactions are reversible  Example: Glucose PO4 Isomerase VI. LIGASES Enzymes causing bond formation between two molecules to form a larger molecule Examples: Ligase Amino Acyl t-RNA synthetase 1. Type of reaction catalyzed 2. Suffix “ASE” added to the name of substrate 3. EMPIRICAL Name Examples: Trypsin, Pepsin 4. STANDARD SYSTEM - Formulated by IUB and IUPAC A. Systematic Name  Describes the nature of the reaction catalyzed  Numerical code designation prefixed w/ the letters E.C.  Examples: E.C. 3.1.3.1 = ALP E.C. 3.1.3.2 = ACP 1st digit = denotes the class of the enzyme 2nd digit = sub-class of the enzyme 3rd digit = subsub-class 4th digit = specific serial number B. TRIVIAL NAME  a.k.a Non- specific, Practical name, Working name  A simplification of the systematic name  Uses acronyms and abbreviations  Examples: SGOT, SGPT Several distinct forms of enzymes Important in the diagnosis of specificity A. ISOENZYMES - Multichained enzymes of similar activity - Tissue/organ & cell organelle- specific Example: Lactate Dehydrogenase (LDH)  Contains H & M sub-units (polypeptide chains) H4 (LD1) – heart, RBC & renal tubules H3M (LD2) – heart, RBC & renal tubules H2M2 (LD3) – Lungs, Lymphocytes, spleen, pancreas HM3 (LD4) – Liver, skeletal muscles M4 (LD5) – Liver, skeletal muscles 1. Electrophoretic mobility 2. Mobility in Ion Exchange Resin 3. Response to Inhibition E.g. ACP (RBC) – inhibited by 2% Formalin ACP (Prostate) – not inhibited by 2% Formalin 4. Relative substrate specificities E.g. ACP (RBC) is less sensitive to α-naphthyl PO4 B. HETEROENZYME - Enzymes of similar catalytic activity but are specie specific E.g. LDH of rabbits & humans C. ALLOENZYME  Genetically-transmitted enzyme  Important in defining the biochemical characteristics of an individual  Forensic medicine & genetics CLASSIFICATION OF ENZYMES IN BLOOD A. PLASMA-SPECIFIC ENZYMES 1. Generally secreted by the liver 2. Enzymes that exert their function in plasma 3. Examples: Coagulation factors Fibrinolytic factors Complement system B. NON-PLASMA SPECIFIC ENZYMES 1. No specific functions in plasma (lack of activators or coenzymes) 2. Two Classes: B.1 Enzymes of Secretion - secreted in plasma at a high rate but rapidly disposed off to excretory channels - low concentration in plasma is maintained  B.2 Enzymes Associated w/ Cellular Metabolism - carry out their functions within the cells in which they are formed - e.g. Creatine kinase = Heart ACP = Prostate gland E + S ES P + E Where: E = unchanged enzyme (catalyst) S = substrate P = transformed substrate ES = postulated enzyme- substrate complex  Energy – activation energy  Molecular Compatibility – commonness between Enzyme & Substrate  Space Availability – # of enzymes or substrates that can be reacted  Specificity – refers to the enzyme acting on a specific substrate Lock & Key – refers to the active site being complementary in shape & size to the substrate Induced Fit Model – the enzyme changes in shape during binding to accommodate the substrate Inhibitors - Decrease the rate of enzyme reaction - Functions: vBinds to the active site, blocks access of the S to the E (Competitive Inhibition) vBinds elsewhere on the E causing change in shape that interferes w/ S binding (Non-Competitive Inhibition) vBinds w/ the E-S complex; no product formed (Uncompetitive Inhibition)  TYPES of INHIBITION a. Reversible Inhibition - Inhibitors are possible removed from the system; enzyme is fully restored - Physical processes that remove inhibitors: dialysis, gel filtration b. Irreversible Inhibition - Inhibitors covalently combine w/ the enzyme - Physical methods are ineffective in separating inhibitors from the enzymes 1. Excess substrate – causes competition between substrate molecules for a single binding site 2. Product of reaction – may be an inhibitor of the forward reaction 3. E-S complex does not break to yield products 4. Chemical substances E.g. Flouride, Oxalate, Heparin, Cupric ion, Tartrate Disruption of the 3-dimensional structure of the enzyme molecule Leads to the loss of enzyme activity May be reversed if  If denaturation is not extensive  If the denaturing agent is removed  Elevated temperature – beyond 50 – 60°C; weakens the stabilizing bonds  Extremes in pH – causes unfolding of enzyme molecule  Radiation  Frothing  Strong salt solution  Mechanical trauma  Time of exposure  Chemicals