CCHM 322 Enzymology (Part 1) - Lecture Notes PDF

DASH CCHM 322 NINE Prelim - Lecture / Roxanne Eushika Medina...

DASH CCHM 322 NINE Prelim - Lecture / Roxanne Eushika Medina Week 01 ENZYMOLOGY (PART I) OUTLINE o heart (cardiac enzymes) 01 Enzymology  disruption of cardiac enzymes to the 02 Enzymes blood vessels causes increased levels of enzymes in the blood 03 Catalytic Mechanism of Enzymes  example, acute myocardial infarction 04 Specificity (CRP) 05 Factors that Influence Enzymatic Reaction o liver (liver enzymes) 06 Enzymatic Reactions  increased in cirrhosis, fibrosis, hepatitis 07 Enzyme Structure (Amino Acids) o muscle (muscle enzymes) 08 Isoenzymes  increased in muscle dystrophy 09 Nomenclature  enzymes + substrate = ES complex = fast chemical reaction  what happens if the cell is damaged? enzymes will ENZYMOLOGY escape from it and go to the circulation that leads to  study of enzymes high concentration of enzymes o clinically significant enzymes can be used in  elevation of enzymes in circulation indicates determining or diagnosing presence of disease problems in cells in the body  catalyze only single reaction o there are thousands of enzymes present in the area where the substrate binds Active site body to enzymes o enzymes are made up of protein that catalyze a Allosteric site area other than the active site chemical reaction o enzymes are a bio-catalase (makes reaction faster) CATALYTIC MECHANISM OF ENZYMES  chemical reaction may occur spontaneously if the free ENZYMES energy or available kinetic energy is higher for the  biological proteins that catalyze a biochemical reactant than the product (lower energy) reaction reactants have enough excess energy to Activation  proteins that serves as a catalase break their bond and collide to form new Energy  all tissues have their specific enzymes bond (enzyme + substrate) EXAMPLES  digestion of food SPECIFICITY  utilization of energy from food  exchange of oxygen and CO2  enzymes are highly specific enzyme combines with only one ESSENTIAL TO PHYSIOLOGIC FUNCTIONING Absolute substrate and catalyzes a single Specificity Hydration of CO2, important for the maintenance of reaction (most strict model)  Group enzyme combines will all substrate with pH in the blood Nerve conduction, some enzymes are found in the Specificity a particular chemical group  Bond enzyme combines with all the substrate brain (CKBB) Muscle contraction, enzymes gives energy to the Specificity with a specific chemical bond  enzyme combines with all the muscles, making contraction possible (CKMM) Stereoisomeric Nutrient degradation in GIT, amylase and lipase for substrates with a specific optical  Specificity the degradation of food isomer  Energy use in GIT IMPORTANT NOTES:  enzymes are located within the cells  specific enzyme for a specific origin  DASH CCHM 322 NINE Prelim - Lecture / Roxanne Eushika Medina Week 01 FACTORS THAT INFLUENCE ENZYMATIC RECTION INHIBITORS SUBSTRATE CONCENTRATION /  interfere ENZYME CONCENTRATION TYPE OF INHIBITION  follows the hypothesis of Michaelis and Menten, “ Even in low substrate concentration, the substrate Competitive inhibitors target the active site can readily bind with free enzyme” Inhibition  enzymatic reaction can be: Non-competitive inhibitors target the allosteric site rate of chemical reactions is Inhibition First-order dependent/directly related (directly Uncompetitive inhibitors target the ES complex Kinetic proportional) to substrate Inhibition concentration rate of chemical reactions is STORAGE Zero-order dependent/directly related (directly Kinetic proportional) to enzyme Freezing temp. long term preservation concentration (-20°C) Refrigerator temp. general temperature (2-8°C) preservation even if we add more reactants, once the Saturation Room temp. for cold labile enzymes (lactate maximum rate of reaction is reached, it will no Kinetics (15 to 25-30°C) dehydrogenase [LD4 & LD5]) longer result in much faster reaction  cold temperature causes reversible inactivation of enzyme PH (POWER OF HYDROGEN)  avoid repeated thawing  optimum: 7.0-8.0 pH  enzymes are protein that carry a net molecular HEMOLYSIS charge (subject to pH, can cause deactivation)  alkalinic and acidic pH may destroy/forfeit some  destruction of RBC enzyme  increased hemolysis = increased K, LDH, ACP, AST and ACP more active at acidic pH (up to 4.0) aldolase, Mg, phosphate (elevated analytes) ALP more active at alkaline pH (up to 10.0) LACTESCENCE / MILKY SPECIMEN TEMPERATURE  decrease in concentration  chylomicrons cause milky specimen  increased temperature = increased reaction  temperature coefficient: increase in every 10°C = 2- ENZYMATIC REACTIONS fold increase on enzyme activity 40-50°C denaturation of enzyme  measured by their activity, not by their absolute value 60-65°C inactivation of enzyme  very small quantity 37°C (25 or (optimum temperature) 30°C [RT]) water bath / body temperature TWO METHODS IN MEASURING THE ENZYMATIC low / cold (refrigerator) temperature reversible activation of enzyme REACTIONS repeated (from frozen to body temperature) end point, single measurement thawing denature enzymes Fixed-time / (reactants are combines allowed to End-point react in designated time reaction assay CO-FACTORS stopped reaction measured) Continuous- multiple measurements, continuous  non-protein entities that is bind to enzymes for a monitoring / measurement/monitoring (measures the reaction to occur Kinetic assay activity of enzymes) made of inorganic factors, alter the spatial configuration enzyme Activators Metallic Non-metallic ENZYME STRUCTURE (AMINO ACIDS) Ca, Fe, Mg, Mn, Zn, K Br, Cl  enzyme is a protein made of organic factors, serve as a second Coenzyme substrate to enzymatic reactions (vitamins, NAD, nucleotide phosphatase)  DASH CCHM 322 NINE Prelim - Lecture / Roxanne Eushika Medina Week 01 Primary made of a specific sequence of amino NOMENCLATURE structure acids  meaning of enzymes developed by the Enzyme Secondary made of polypeptide chain twisting Commission (E.C.) structure Oxido- oxidation-reduction reaction between two Tertiary made of folding of secondary structure reductase substrates structure (globular) transfer of group other than hydrogen Quaternary combination of tertiary structure Transferase from one substrate to another structure (complex) Hydrolase hydrolysis of various bonds removal of group from substrate without ISOENZYMES Lyase hydrolysis (products contain double  enzymes with same catalytic function but different bonds) physical properties interconversion of geometric, optical, or Isomerase  differentiated by: positional isomers  electrophoretic mobility joining of two substrate molecules,  solubility coupled with breaking of the Ligase  resistance to inactivation pyrophosphate bond in adenosine triphosphate or a similar compound NOTE: Creatine kinase (3 isoenzymes) IMPORTANT NOTES: CK1 - CKBB brain (anodal)  1st number: class CK2 heart  2nd number: subclass CK3 - CKMM muscle (least anodal)  3rd number: sub-subclass  Final number - serial number CLASSIFICATION OF FREQUENTLY QUANTITATED ENZYMES RECOMMENDED COMMON STANDARD CLASS EC CODE NO. SYSTEMATIC NAME NAME ABBREVIATION ABBREVIATION Lactate L-Lactate: NAD+ LDH LDH 1.1.1.27 dehydrogenase oxidoreductase Glucose-6- D-Glucose-6- phosphate G-6-PDH G-6-PD 1.1.1.49 Oxidoreductase phosphate: NADP dehydrogenase L-glutamate: NAD(P) Glutamate GLD GLD 1.4.1.3 oxidoreductase, dehydrogenase deaminase GOT (glutamate L-Aspartate: 2- Aspartate oxaloacetate AST 2.6.1.1 oxaglutarate aminotransferase transaminase) aminotransferase L-Alanine: 2- Alanine GPT (glutamate ALT 2.6.1.2 oxaloglutarate aminotransferase transaminase) aminotransferase CPK (creatine ATOL creatine N- Creatine kinase CK 2.7.3.2 phosphokinase) phosphotransferase (5-Glutamyl) Transferase y-Glutamyl- peptide: amino acid- GGPT GGT 2.3.2.2 transferase 5- glutamyltransferase Glutathione-S- Glutathione a-GST GST 2.4.1.18 transferase transferase 1,3-a-D-Glucan: Glycogen orthophosphate-a- GP GP 2.4.1.1 phosphorylase D- glucosyltransferase Pyruvate kinase PK PK 2.7.1.40 Pyruvate kinase  DASH CCHM 322 NINE Prelim - Lecture / Roxanne Eushika Medina Week 01 Orthophosphoric Alkaline monoester ALP ALP 3.1.3.1 phosphatase phosphohydrolase (alkaline optimum) Orthophosphoric monoester Acid phosphatase ACP ACP 3.1.3.2 phosphohydrolase (acid optimum) Hydrolase 1,2-D-Glucan a-Amylase AMY AMS 3.2.1.1 gluconohydrolase Chymotrypsin CHY CHY 3.1.21.1 Chymotrypsin Elastase-1 E1 E1 3.1.21.36 Elastase 5’-Ribonucleotide 5-Nucleotidase NTP NTP 3.1.3.5 phosphohydrolase Triacylglycerol Triacylglycerol TPS 3.1.1.3 lipase acylhydrolase Trypsin TRY TRY 3.4.21.4 Trypsin D-D-Fructose-1,6- bisdiphosphateD- Lyase Aldolase ALD ALD 4.1.2.13 glyceraldehyde-3- phosphate-lyase Triosephosphate 6 Triose-phosphate Isomerases TPI TPI 5.3.1.1 isomerase isomerase Glutathione Glutathione Ligase GSH-S GSH-S 6.3.2.3 synthase synthase