Enzymes Part 1 M. Zaharna Clin. Chem. 2009

Introduction Enzymes are usually proteins that act as catalysts, compounds that increase the rate of chemical reactions. They bind specifically to a substrate, forming a complex. This complex lowers the activation energy in the reaction: without the enzyme becoming consumed and without changing the equilibrium of the reaction. A product is produced at the end of the reaction M. Zaharna Clin. Chem. 2009

Introduction The catalyzed reactions are frequently specific and essential to physiologic functions, such as: the hydration of carbon dioxide, nerve conduction, muscle contraction, nutrient degradation, and energy use. Found in all body tissue, enzymes frequently appear in the serum following cellular injury or, sometimes, in smaller amounts, from degraded cells. M. Zaharna Clin. Chem. 2009

M. Zaharna Clin. Chem. 2009

General Properties of Enzymes Like all proteins 1°, 2°, 3°, and 4° structures Active site → cavity where substrate interacts Often water-free site Reacts with charged amino acid residues Allosteric site Another site on enzyme where co-factors or regulatory molecules interact M. Zaharna Clin. Chem. 2009

Isoenzymes Isoenzymes – are enzymes that differ in amino acid sequence but catalyze the same chemical reaction. They have similar catalytic activity, but are different biochemically or immunologically. Different forms may be differentiated from each other based on certain physical properties electrophoretic mobility, differences in absorption properties or by their reaction with a specific antibody These enzymes usually display different kinetic parameters (i.e. different KM values), or different regulatory properties. The existence of isozymes permits the fine-tuning of metabolism to meet the particular needs of a given tissue or developmental stage M. Zaharna Clin. Chem. 2009

Cofactors Non-protein molecules required for enzyme activation Inorganic Activators Chloride or magnesium ions, etc. Organic Coenzymes e.g. Nicotinamide adenine dinucleotide (NAD) M. Zaharna Clin. Chem. 2009

Classes of Enzymes International Union of Biochemistry (IUB) 1 = Oxidoreductases (Examples: LDH, G6PD) Involved in oxidation - reduction reactions 2 = Transferases (Examples: AST, ALT) Transfer functional groups 3 = Hydrolases (Examples: acid phosphatase, lipase) Transfer groups to -OH 4 = Lyases (Examples: aldolase, decarboxylases) Add across a double bond 5 = Isomerases (Example: glucose phosphate isomerase) Involved in molecular rearrangements 6 = Ligases Complicated reactions with ATP cleavage Catalyze the joining of two substrate molecules M. Zaharna Clin. Chem. 2009

M. Zaharna Clin. Chem. 2009

Enzyme classification Plasma vs. non-plasma specific enzymes Plasma specific enzymes have a very definite/specific function in the plasma 1) Plasma is normal site of action 2) Concentration in plasma is greater than in most tissues 3) Often are liver synthesized 4) Examples: cholinesterase, plasmin, thrombin cholinesterase is an enzyme that catalyzes the hydrolysis of the neurotransmitter acetylcholine into choline and acetic acid, a reaction necessary to allow a cholinergic neuron to return to its resting state after activation. M. Zaharna Clin. Chem. 2009

Enzyme classification Non-plasma specific enzymes have no known physiological function in the plasma 1) Some are secreted into the plasma 2) A number of enzymes associated with cell metabolism normally found in the plasma only in low concentrations. An increased plasma concentration of these enzymes is associated with cell disruption or death M. Zaharna Clin. Chem. 2009

Factors Affecting Enzyme Levels in Blood Entry of enzymes into the blood Leakage from cells Altered production of enzymes E.g. increased osteoblastic activity results in increase in enzymes in bone disease Clearance of enzymes Half life vary from few hours to several days M. Zaharna Clin. Chem. 2009

Factors That Influence Enzymatic Reactions Substrate Concentration Enzyme Concentration pH Temperature Cofactors Inhibitors M. Zaharna Clin. Chem. 2009

Measuring enzyme activity Enzymes are usually present in very small quantities in biologic fluids and often difficult to isolate from similar compounds Therefore, Enzymes are not directly measured Enzymes are commonly measured in terms of their catalytic activity We don’t measure the molecule … But we measure how much “work” it performs (catalytic activity) That means the rate at which it catalyzes the conversion of substrate to product The enzymatic activity is a reflection of its concentration Activity is proportional to concentration M. Zaharna Clin. Chem. 2009

Measuring enzyme activity Enzyme activity can be tested by measuring Increase of product Decrease of substrate Decrease of co-enzyme Increase of altered co-enzyme If substrate and co-enzyme are in excess concentration, the reaction rate is controlled by the enzyme activity. M. Zaharna Clin. Chem. 2009

Measuring enzyme activity NADH ( a common co-enzyme ) ( the reduced form ) absorbs light at 340 NM NAD does not absorb light at 340 nm Increased ( or decreased ) NADH concentration in a solution will cause the Absorbance ( A ) to change. M. Zaharna Clin. Chem. 2009

Measurement Conditions Excess amounts of substrate and any cofactors or coenzymes to handle possible abnormally high patient enzyme levels Proper temperature and pH Inhibitors must be lacking The temperature should be constant within ±0.1°C throughout the assay at a temperature at which the enzyme is active M. Zaharna Clin. Chem. 2009

Methods for Enzyme measurement Fixed time methods the reactants are combined, the reaction proceeds for a designated time, the reaction is stopped (usually by inactivating the enzyme with a weak acid), a measurement is made of the amount of reaction that has occurred. The reaction is assumed to be linear over the reaction time; the larger the reaction, the more enzyme is present. Possible problems with extremely high enzyme levels M. Zaharna Clin. Chem. 2009

Methods for Enzyme measurement Continuous-monitoring methods multiple measurements, usually of absorbance change, are made during the reaction, either at specific time intervals (usually every 30 or 60 seconds) or continuously by a continuous-recording spectrophotometer. These assays are advantageous over fixed-time methods because the linearity of the reaction may be more adequately verified. M. Zaharna Clin. Chem. 2009

Methods for Enzyme measurement Continuous-monitoring methods If absorbance is measured at intervals, several data points are necessary to increase the accuracy of linearity assessment. Continuous measurements are preferred because any deviation from linearity is readily observable. M. Zaharna Clin. Chem. 2009

Measurement Units Reported as “activity” not concentration IU = amount of enzyme that will convert 1 μmol of substrate per minute in specified conditions Usually reported in IU per liter (IU / L) SI unit = Katal = mol/sec moles of substrate converted per second enzyme reported as katals per liter (kat / L) 1 IU = 17nkat International System of Units M. Zaharna Clin. Chem. 2009

Measurement of Enzyme Mass Immunoassay methodologies that quantify enzyme concentration by mass are also available and are routinely used for quantification of some enzymes. Immunoassays may overestimate active enzyme as a result of: possible cross-reactivity with inactive enzymes, inactive isoenzymes, or partially digested enzyme. M. Zaharna Clin. Chem. 2009

Measurement of Enzyme Mass The relationship between enzyme activity and enzyme quantity is generally linear but should be determined for each enzyme. Enzymes may also be determined and quantified by electrophoresis techniques which provide resolution of isoenzymes. M. Zaharna Clin. Chem. 2009

M. Zaharna Clin. Chem. 2009

Creatine Kinase (CK) Action – This enzyme is associated with the regeneration and storage of high energy phosphate (ATP). It catalyzes the following reversible reaction in the body. M. Zaharna Clin. Chem. 2009

Creatine Kinase (CK) High concentrations of CK in: skeletal muscle, cardiac muscle and brain tissue Increased plasma CK activity is associated with damage to these tissues  CK is especially useful to diagnose: Acute Myocardial Infarction (AMI) Skeletal muscle diseases ( Muscular Dystrophy ) Muscular dystrophy (MD) refers to a group of genetic, hereditary muscle diseases that weaken the muscles that move the human body.[1][2] Muscular dystrophies are characterized by progressive skeletal muscle weakness, defects in muscle proteins, and the death of muscle cells and tissue M. Zaharna Clin. Chem. 2009

Creatine Kinase (CK) CK has 3 isoenzymes Each isoenzyme is composed of two different polypeptide chains (M & B) CK - BB (CK1) Brain type CK - MB (CK2) Cardiac type or hybrid type CK – MM (CK3) Muscle type Normal serum consists of approximately 94% to 100% CK-MM Cardiac muscle CK is 80% CK-MM and 20% CK-MB M. Zaharna Clin. Chem. 2009

Creatine Kinase (CK) BB migrates fastest to anode than MB & MM The major isoenzyme in the sera of healthy people is the MM form. Values for the MB isoenzyme range from undetectable to trace (<6% of total CK). It also appears that CK-BB is present in small quantities in the sera of healthy people M. Zaharna Clin. Chem. 2009

Diagnostic Significance The value of CK isoenzyme separation can be found principally in detection of myocardial damage. Cardiac tissue contains significant quantities of CK-MB, approximately 20% of all CK-MB. increased CK – MB ( > 6% of the total CK activity ) is a strong indication of AMI Post AMI CK-MB CK-MB increases 4 – 8 hours post AMI Peaks at 12 - 24 hours post AMI Returns to normal 48 - 72 hours M. Zaharna Clin. Chem. 2009

M. Zaharna Clin. Chem. 2009

CK Assay CK assays are often coupled assays. In the example below, the rate at which NADPH is produced is a function of CK activity in the first reaction. Hexokinase and G6PD are auxiliary enzymes Reverse reaction most commonly performed in clinical laboratory methods M. Zaharna Clin. Chem. 2009

CK Assay Reference Range for Total CK: Male, 15-160 U/L (37°C) Female, 15-130 U/L (37°C) CK-MB: <6% total CK M. Zaharna Clin. Chem. 2009

CK isoenzymes For CK isoenzymes, electrophoresis is the reference method. Other methods include ion-exchange chromatography, and radioimmunoassay. Rapid assay for CK-MB subforms, uses high voltage electrophoresis on an automated analyzer, the result will be available in 25 min. M. Zaharna Clin. Chem. 2009

Lactate Dehydrogenase (LD) Catalyzes interconversion of lactic and pyruvic acids It is a hydrogen-transfer enzyme NAD is used as coenzyme High activities in heart, liver, muscle, kidney, and RBC Lesser amounts: Lung, smooth muscle and brain M. Zaharna Clin. Chem. 2009

LDH Isoenzymes Because increased total LDH is relatively non-specific, LDH isoenzymes can be useful 5 isoenzymes composed of a cardiac (H) and muscle ( M ) component LD - 1 ( HHHH ) Cardiac , RBCs LD - 2 ( HHHM ) Cardiac , RBCs LD - 3 ( HHMM ) Lung, spleen, pancreas LD - 4 ( HMMM ) Hepatic LD - 5 ( MMMM ) Skeletal muscle LD-1 is the fastest towards the anode M. Zaharna Clin. Chem. 2009

Diagnostic Significance LDH is elevated in a variety of disorders. in cardiac, hepatic, skeletal muscle, and renal diseases, as well as in several hematologic and neoplastic disorders The highest levels of LD-1 are seen in pernicious anemia and hemolytic disorders LD-3 with pulmonary involvement LD-5 predominates with liver & muscle damage M. Zaharna Clin. Chem. 2009

Diagnostic Significance In healthy individuals LD-2 is in highest quantity then LD-1, LD-3, LD-4 and LD-5 Heart problems: 2-10 x (Upper Limit of Normal) ULN in acute MI If problem is not MI, both LD1 and LD2 rise, with LD2 being greater than LD1 If problem is MI, LD1 is greater than LD2. This is known as a flipped pattern M. Zaharna Clin. Chem. 2009

Diagnostic Significance A sixth LDH isoenzyme has been identified LDH-6 has been present in patients with arteriosclerotic cardiovascular failure Its appearance signifies a grave prognosis and impending death It is suggested, that LDH-6 may reflect liver injury secondary to severe circulatory insufficiency M. Zaharna Clin. Chem. 2009

Assay for Enzyme activity The reaction can proceed in either a forward or reverse direction Pyruvate + NAD+ Lactate + NADH + H+ The optimal pH: for the forward reaction is 8.3 – 8.9 For the reverse reaction 7.1 – 7.4 Reference Range : 100-225 U/L (37°C) LD M. Zaharna Clin. Chem. 2009

Aspartate Aminotransferase (AST, SGOT, GOT) Transferase class of enzymes - transaminase Transaminase involved in the transfer of an amino group between aspartate and -ketoacids. Pyridoxal phosphate is coenzyme Source is heart, liver, and skeletal muscle Serum Glutamic Oxalocetic Transaminase – SGOT The older terminology, serum glutamic-oxaloacetic transaminase (SGOT, or GOT) M. Zaharna Clin. Chem. 2009

Aspartate Aminotransferase (AST, SGOT, GOT) The transamination reaction is important in intermediary metabolism because of its function in the synthesis and degradation of amino acids. The ketoacids formed by the reaction are ultimately oxidized by the tricarboxylic acid cycle to provide a source of energy. M. Zaharna Clin. Chem. 2009

Diagnostic Significance The clinical use of AST is limited mainly to the evaluation of hepatocellular disorders and skeletal muscle involvement. Post AMI Rises 6 – 8 hours Peaks at 24 hours Returns to normal by day 5 AST levels are highest in acute hepatocellular disorders, viral hepatitis, cirrhosis. Viral hepatitis may reach 100 x ULN M. Zaharna Clin. Chem. 2009

Diagnostic Significance There are two isoenzyme fractions located in the cell cytoplasm and mitochondria, the cytoplasmic isoenzyme is predominant in serum while the mitochondrial one may be increased following cell necrosis. Isoenzyme analysis of AST is not routinely performed in the clinical laboratory. M. Zaharna Clin. Chem. 2009

Assay for Enzyme activity Measurement by Karmen method – use Malate dehydrogenase in second step Detect change in absorbance at 340 nm Aspartate + -Ketoglutarate Oxaloacetate + Glutamate Oxaloacetate + NADH + H Malate + NAD MD AST Reference Range : 5 to 30 U/L (37°C) M. Zaharna Clin. Chem. 2009