Enzymes - Part 1 PDF

CHEM2010 Enzymes - PART 1 (Liver, Pancreas, & Cardiac) CHAPTER 13 – TEXTBOOK (8TH EDITION) CHAPTER 8 - TEXTBOOK (9TH EDITION)  Enzymes Are specific biologic proteins that catalyze biochemical reactions ◦ i.e. they lower the activation energy needed for the reactio...

CHEM2010 Enzymes - PART 1 (Liver, Pancreas, & Cardiac) CHAPTER 13 – TEXTBOOK (8TH EDITION) CHAPTER 8 - TEXTBOOK (9TH EDITION)  Enzymes Are specific biologic proteins that catalyze biochemical reactions ◦ i.e. they lower the activation energy needed for the reaction to proceed They are not consumed or changed in composition ◦ Enzymes are regenerated and act upon a substrate. Can appear in all body tissues and frequently appear in serum following cellular injury or degradation. They are proteins, comprising specific amino acid sequences. ◦ Therefore, they can be denatured by heat, changes in pH, etc. They may exist in different forms – isoenzymes and isoforms  Enzyme Nomenclature The Enzyme Commission (EC) developed a classification system in 1961. It includes: Systematic Name – defines substrates acted on, reaction catalyzed & name of any coenzymes Recommended Name – more usable / practical Abbreviation – widely used in laboratories Code Number – four digit number separated by decimal points - 1st digit places enzymes into one of six classes.  Classes of Enzymes The International Union of Biochemistry (IUB) system assigns a name and code to each enzyme. 1. Oxidoreuctases catalyze an oxidation-reduction reaction between 2 substrates. 2. Transferases catalyze transfer of a group other than hydrogen from one substrate to another. 3. Hydrolases catalyze hydrolysis of various bonds. (H2O added/substrate broken down) 4. Lyases catalyze removal (-lyse) of groups from substrates without hydrolysis; End product will contain double bonds 5. Isomerases catalyze interconversion of geometric, optical, or positional isomers - ex. Convert D to L forms 6. Ligases catalyze joining (-ligate) of two substrate molecules coupled with breaking of pyrophosphate bond in ATP  This semester we will be focusing on: - LDH – Lactate dehydrogenase - AST – Aspartate aminotransferase ** Educational purposes only ** - ALT – Alanine aminotransferase - CK – Creatine kinase - GGT – γ-Glutamyltransferase - ALP – Alkaline phosphatase - ACP - Acid phosphatase - AMY - Amylase  Measurement of Enzyme Activity It is common to measure the activity of an enzyme and relate that to its concentration. Enzyme concentrations in blood are normally very low. ◦ Therefore, increased levels in blood indicate a disease process. Immunoassays can be used to detect some enzymes directly. ◦ Measure enzyme concentration by mass (e.g. Creatine Kinase, CK) Electrophoretic techniques ◦ Used to measure isoenzymes or isoforms Measurement of Enzyme Activity  Enzyme Kinetics There are 3 phases: 1. Lag Phase - Sample and reagents are mixed - Equilibrium is established - Non-linear 2. Linear Phase - Product formation and substrate consumption are consistent - Follows zero order kinetics - This is where we measure 3. Substrate Depletion - Product formation has slowed - Not linear - Not good for measuring - Problem if enzyme concentration is very high Enzyme Kinetics If enzyme concentration is very high in a sample the reaction will not be linear as you will run out of substrate. The sample can be diluted and reanalyzed.  Methods for Reading Enzyme Reactions 2-Point Assay (Fixed time) ◦Read at fixed times ◦Usually an initial reading and a later reading ◦Readings may not be during linear phase (problem) Kinetic Assay (Continuous Monitoring) ◦Multiple absorbance readings taken as the reaction proceeds. (Every 30-60 sec or continuously) ◦More accurate ◦Deviations from linearity will be detected.  Zero Order Kinetics Exist during the linear phase The rate of reaction is only dependent on enzyme concentration An increase in substrate will not increase the reaction rate Lab enzyme procedures follow zero order kinetics If other variables effect rate (e.g. substrate depletion), then First-Order kinetics exist If we are trying to measure [enzyme] then we must use excess substrate. If there is not enough substrate, the enzyme will have nothing to act upon. Michaelis-Menton Curve Km = Michaelis constant = [substrate] @ ½ max velocity Manufacturers ensure the [substrate] in enzyme assay kits are 10-100X the Km to prevent substrate depletion. Zero Order Kinetics Substrate in excess  Factors the Influence Enzyme Activity Substrate Concentration – make sure that substrate does not run out pH - enzymes have an optimal pH - reactions should be buffered at the optimal pH - changes in pH may denature the enzyme - most physiologic enzymatic reactions occur in the pH range of 7.0 to 8.0 Temperature - increasing the temperature will increase the enzyme activity (to a point!) - if the temperature is increased too high the enzyme will be denatured - rate of reaction doubles for each 10 degree increase, Q10 - usual temps are 25, 30 & 37 ± 0.1oC Enzyme Concentration - the higher the enzyme concentration, the faster the reaction will proceed.  Cofactors Nonproteins Bind to an enzyme before a reaction can occur Examples: Activators ◦ Inorganic cofactors ◦ Metals (Ca2+, Fe2+, Mg2+) ◦ Nonmetals (Br-, Cl-) Coenzymes ◦ Organic cofactors ◦ Phosphates, vitamins  Inhibitors and Activators Activators - cause rate of reaction to increase - inorganic cofactors - required for reaction - often metal ions (e.g. Ca2+, Fe2+, Mg2+, Mn2+, Zn2+, K+) or nonmetallic ions (e.g. Br- and Cl-) Inhibitors - cause rate of reaction to decrease - interfere - may be reversible or irreversible  Enzyme Terms Coenzyme - organic cofactor - e.g. vitamins, NAD - not tightly bound Prosthetic group - tightly bound coenzyme Holoenzyme - active enzyme Apoenzyme - inactive enzyme - only the enzyme portion; no coenzyme Proenzyme or Zymogen - structurally inactive form - other enzymes later alter the structure to make it active - mostly digestive enzymes - e.g. Trypsinogen → Trypsin (proenzyme) (active enzyme)  How Do We Report Enzyme Activity? Historically different units were used for different methods Currently the International Unit is used: ◦Defined as the amount of enzyme that will catalyze the reaction of 1 μmol of substrate per minute per liter of serum ◦Written as IU/L or mIU/mL  Enzymes as Reagents Enzymes can be used as reagents to measure nonenzymatic analytes in serum ◦Example: Glucose oxidase can be used when measuring glucose Uricase can be used when measuring uric acid The rate of reaction is proportional to the substrate concentration.  Enzyme Analysis vs. Enzymes as Reagents To measure enzymes: - Zero order - Substrate must be in excess - Enzyme is rate limiting factor - So Rate ~ [enzyme] Using enzyme as a reagent to measure another substrate: - First order - Enzyme is in excess - Substrate is rate limiting factor Using enzymes to measure - The more substrate the faster it will go something else (glucose, uric acid) - So Rate ~ [substrate]  Why Measure Enzymes? Enzymes are produced within cells and found in all body tissues. Those that are found in the blood are either: ◦Plasma specific (have a function in the bloodstream) E.g. Coagulation enzymes (fibrinolytic enzymes) No known function in blood ◦Secreted ◦Cellular An increase in secreted or cellular enzymes indicates disease or damage of the organ(s). ◦Disease can cause ↑’d production from the tissue or organ ◦Damage - causes leakage of enzymes into bloodstream  Isoenzymes Isoenzymes = Different forms of the same enzyme; differ in amino acid sequence but catalyze the same reaction. Have the same active site → Can catalyze the same reaction but have different structures ◦e.g. ALP (from different organs) always catalyzes the breakage of organic phosphate esters Come from different organ/tissues ◦ e.g. ALP isoenzymes come from intestine, liver, bone, spleen, placenta, & kidney Why do we identify isoenzymes? ◦ To tell us which organ is involved when there is more than one source.  Methods to ID Isoenzymes Zone Electrophoresis (AKA protein electrophoresis) ◦Enzymes have different protein structures ◦After electrophoresis we get visible bands after exposure to specific chemicals Selective Inactivation Temp & chemicals can denature or inhibit certain ◦Increase temperatureisoenzymes while leaving others intact ◦Chemical Inactivation ◦Immunochemical - uses antisera to ID specific isoenzymes Clinically Significant Enzymes in Med Lab Sciences (Chemistry)  Clinically Significant Enzymes in Med Lab Alkaline Phosphatase (ALP) Acid Phosphatase (ACP) Amylase (AMS) Aspartate Transaminase (AST) Alanine Aminotransferase (ALT) Gamma Glutamyltransferase (GGT) Lactate Dehydrogenase (LD) Creatine Kinase (CK) Alkaline Phosphatase (ALP) Alkaline Phosphatase (ALP) Group specificity - acts on phosphate ester structure  Isoenzymes of Alkaline Phosphatase (ALP) Four major isoenzymes: ◦ Bone, Liver, Intestine, and Placenta Others: Regan, Nagao, Pa, fast liver → associated with neoplasms ◦ Fast liver found in metastatic liver cancer To differentiate between isoenzymes: ◦ We use electrophoresis: Anode Liver Bone/Placental Intestine Cathode + X X X - ◦ Use heat → Will determine if bone or placental (placental is the most heat stabile) ◦ Use selective chemical inhibition - Urea will inactivate the isoenzymes (bone 1st) ◦ Immunochemical methods – Use antibodies specific to placenta, bone, etc. Alkaline Phosphatase (ALP) - Clinical Significance Evaluation of liver and bone disorders/disease Increase in ALP seen in: ◦Extrahepatic obstruction (3-10X normal) ◦Intrahepatic obstruction ( 50 years old ◦3rd Trimester of Pregnancy - increase in placental ALP ◦ Increase in ALP also seen with complications ◦ Returns to normal 3-6 days after delivery Decrease in ALP seen in: ◦ Hypophosphatasia - inherited disorder with absence of bone isoenzyme ◦ Results in inadequate bone calcification Alkaline Phosphatase (ALP) - Sources of Error Text p. 277 Avoid hemolyzed samples (ALP is 6X higher in RBCs) Analyze ASAP (3-10% increase on standing) Group O and B secretors (25% higher after a high-fat meal) Acid Phosphatase (ACP / PAP) Acid Phosphatase (ACP) Group specificity - acts on phosphate ester structure Isoenzymes of Acid Phosphatase (ACP) ACP found in liver, bone, spleen, and kidney but highest in prostate (!). Can differentiate prostatic ACP (PAP) from others by: ◦ Earlier manual methods: tartrate added to inhibit prostatic form. ◦ Total ACP - ACP after tartrate inhibition = PAP ◦Newer methods are based on the fact that PAP reacts faster than others (breaks down substrate faster). So, timing can be used to measure PAP activity.  Acid Phosphatase - Clinical Significance Evaluation of prostate carcinoma and used in forensic investigations ACP increased in: ◦Metastatic prostate carcinoma (50X normal) ◦ Useful to confirm and stage metastatic prostate cancer ◦ Local cancer will show only slight increase or normal values ◦Vaginal washings after rape ◦ Can persist up to 4 days following rape ◦ Useful in forensic investigations ◦Bone Disease ◦Platelet Damage  Acid Phosphatase (ACP) - Sources of Error Red blood cells and platelets have ACP ◦Separate serum from cells ASAP to avoid a false increase ACP is VERY UNSTABLE at room temperature. If not tested immediately, freeze or acidify to pH < 6.5 ◦Loss of CO2 = ↑ pH = ↓ ACP ◦So need to acidify to pH