Enzyme Reaction Kinetics & Clinical Enzymology

History of Enzymes

• Alpha-amylase was identified to break down starch more efficiently than chemical methods in 1835.
• The word "enzyme" comes from the Greek words "en" (in) + "zyme" (yeast).
• This is because initial research studies focused on sugar fermentation investigation in yeast.

Basic Principles of Enzymes

• Enzymes consist of proteins with primary, secondary, tertiary, and quaternary structures.
• Enzymes can trigger an immune response when injected into another species.
• Immunoassay testing uses antigenic properties to test for isoenzymes and isoforms
• Enzymes participate in biological metabolic processes.
• Enzymes are used to construct clinical lab reagents for Spectrophotometry-based testing.
• Enzymes are used to construct clinical lab reagents for Labeled-enzyme Immunoassays, in some formats.

Enzyme Classification

• Enzymes are categorized into 6 main groups: Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases and Ligases.
• Oxidoreductases catalyze oxidation-reduction reactions (hydrogen transfer, addition or removal)
• Transferases catalyze the transfer of functional groups other than hydrogen.
• Hydrolases use water (H₂O) to break a chemical bond.
• Lyases catalyze group elimination to generate double bonds, without hydrolysis (H₂O)
• Isomerases catalyze interconversion of isomers.
• Ligases catalyze bond formation coupled with ATP hydrolysis.

Definitions of Isoenzymes and Isoforms

• Enzymes may exist as isoezymes and isoforms.
• Isoenzymes are defined at the genetic level, which confers different tissue/organ expression.
• Isoenzymes catalyze the same reaction, same substrate to product formation.
• Isoforms are non-genetic modifications of enzymes.
• Isoforms are described as “post-translational” modifications, with slight change or functional group added to the enzyme after protein synthesis.

Enzyme Kinetics

• The enzyme binds the substrate to produce an enzyme-substrate (ES) complex in the formula E + S → ES → P + E.
• The product (P) is formed and then the enzyme (E) separates afterwards from the complex.
• As the enzyme is not depleted, it can catalyze additional reactions.
• Enzymes elevate the speed of metabolic reactions and life needs enzyme involvement.
• Activation energy is how much energy is needed to energize 1 mole of substrate to the activated E-S complex.
• Enzymes lower the energy barrier needed for a reaction to take place.
• V-max represents its maximum velocity when the substrate concentration is high enough that all enzyme molecules are filled and all active sites are engaged.
• The Michaelis-Menten Constant (Km) is equals substrate molar concentration [S] when the reaction velocity is at 1/2 V-max.
• As substrate concentration increases, reaction velocity increases up to a saturation point, until it plateaus and a straight line is formed.
• First order kinetics occurs when reaction velocity is directly proportional to substrate concentration [S]
• Zero order kinetics occurs at enzyme saturation point, where all sites on enzyme are occupied so that no more ES complex formation can take place

Factors Influencing Enzyme Activity

• Defining the following parameters bases Michaelis-Menten kinetics:
• Substrate concentration.
• Temperature.
• Buffer conditions, including pH and salt concentration.
• Cofactors (e.g., B-vitamins, minerals).
• Conditions will be standardized in clinical test assays to ensure consistent results.

Enzyme Inhibitors

• The inhibitors of enzymes have serious effects.
• Enzyme inhibitors may cause harmful or even fatal metabolic disorders.
• Inhibitors typically include drugs and toxic metals, ex, lead (Pb), which inhibits several heme synthesis enzymes needed for heme production, which is used by heme-containing enzymes (e.g., catalase, cytochrome P450), and hemoglobin for oxygen transport.
• Enzyme inhibition can sometimes be reversed by adding more substrate to a reaction to increase its speed, but there are times when that will not be the case as it's irreversible.
• When approaching V-max, it may be difficult to determine the effects of an enzyme inhibitor on the reaction rate since there is non-linear "zero order kinetics" in the M-M graph
• To make more visual, and easier to describe, the M-M equation equation is re-arranged by taking the reciprocal (1/x) and then graphs those data, called a “Lineweaver-Burk plot.”

Clinical Application - Disease correlation/tissue location

• Enzymes play a vital role across many different organs and cells within the body.
• Enzymes are found in muscle, liver, pancreas, heart, bone, erythrocytes and lymphocytes
• Release of cellular enzymes into blood suggests a pathological process like cellular destruction.

Enzymes of Clinical Interest in Blood, their tissue sources and clinical interpretation.

• Alanine aminotransferase (ALT): Liver/Hepatic parenchymal disease.
• Alkaline phosphatase (ALP): Liver, bone, intestinal mucosa, placenta/Hepatobiliary disease and bone disease.
• Amylase: Salivary glands, pancreas/Pancreatic disease (pancreatic isoenzyme).
• Aspartate aminotransferase (AST): Heart, liver, skeletal muscle, erythrocytes/Hepatic parenchymal disease.
• Creatine kinase (CK): Skeletal muscle, heart/Muscle disease.
• γ-Glutamyltransferase (GGT): Liver, pancreas, kidney/Hepatobiliary disease.
• Lactate dehydrogenase (LD): Heart, erythrocytes, lymph nodes, skeletal muscle, liver/Hemolytic and megaloblastic anemias, leukemia, and lymphoma, oncology.
• Lipase: Pancreas/Pancreatic disease.

Enzyme Coenzymes/Cofactors

• Enzymes need these to reach V-max (maximal reaction velocity) and are extremely important in reaction kinetics.
• Play a principal role in metabolism.
• The addition of coenzymes and cofactors to test reagents ensures Vmax is achieved and that lower enzyme values on patients are not reported.
• Enzyme test result with and without the coenzyme demonstrates this effect, termed as a "negative bias"
• The addition of these to test reagent is most essential with patients that have deficient intakes of one or more of these.

Examples of Coenzymes/Cofactors

• Coenzyme is an organic molecule that contain Carbon. Examples are Coenzyme C, Thiamine pyrophosphate, Folic acid coenzymes,Cobamide [B12] coenzymes, Nicotinamide coenzymes, Flavin coenzymes, Biotin, Lipoic acid, Pyridoxal phosphate and Coenzyme Q
• Cofactor is the same as a coenzyme, but is present as an inorganic mineral or metal. Examples are Mg+, Fe++/Fe+++*, Zn++, Cu+/Cu++, Ca++, Mn+ and Co++.++
• B vitamins and inorganic minerals are essential nutrients to prevent a wide range of diseases, which humans have lost the ability to synthesize most of.
• Many B vitamins are coenzymes (NAD+/NADH, as example).

Reporting Units for Enzymes Measurements

• The catalytic rate of an enzyme are expressed in International Unit (IU).
• It is expressed in liter (L) of blood and commonly written as U/L or IU/L.
• US labs primarily measure the enzyme count.
• The international unit (IU) is the amount of enzyme that catalyzes 1 umol of substrate to product per minute under standardized conditions of: temperature, pH, substrate, and coenzymes/cofactors.
• • umol = micromole

Enzymes of Diagnostic Use - Creatine kinase (CK)

• It is a muscle cell damage indicator, but also a marker of acute myocardial infarction (heart attack), although this test is no longer recommended as a primary marker to diagnose AMI.
• Elevated CK suggests muscular disorders, like rhabdomyolysis and muscular dystrophy and severe trauma
• Newer markers like cardiac troponin have since replaced CK.
• Inorganic magnesium (Mg2+) is a cofactor, driving both forward and reverse reactions.
• PH drives the reaction in the "forward direction” or “reverse direction.
• The CK reverse reaction proceeds two to six times quicker than the forward reaction.
• CK → Hexokinase → G-6-Phosphate Dehydrogenase happens at pH 6.7.
• Normal Ranges of Total CK
• Males: 46-300 U/L.
• Females: 15-180 U/L.
• It is affected by age, race, muscle mass and level of physical activity.
• CK enzyme has 3 isoenzymes: (BB, MB, MM)
• B = brain and M=muscle; the combined catalytic activity of all isoenzymes= total activity.
• Of clinical interest, CK-MB is the only isoenzyme still in clinical use.
• Normal Ref. Range is Male < 6.70 ng/mL and Female < 3.80 ng/mL.

Hepatic Enzymes ("Liver function tests" aka LFTs)

• Enzymes' markers levels, increase in blood when there is liver cell destruction or blockage of bile flow.
• Used to diagnose hepatitis, cirrhosis, liver, cancer, fatty liver disease, and bile tract or bile duct obstruction.
• Liver function tests measure enzymes as indicators of liver damage due to hepatocyte destruction or biliary tract disease.
• Liver function tests include AST, ALT, GGT, and ALP (Transaminase enzymes).
• Transaminase enzymes catalyze the interconversion of amino acids and a-ketoacids.
• Transamination used in urea cycle (nitrogen metabolism), products of amino acid metabolism and gluconeogenesis
• Vitamin B-6 is required for Vitamin B-6 activity.
• AST, ALT are the clinically important transaminases: AST (cytoplasmic and mitochondrial forms) and ALT (cytoplasmic form only)
• Cytoplasm contains AST, ALT, LDH while mitochondria has AST, and Membrane has ALK (ALP), GGT.
• Malate dehydrogenase is the indicator reaction measuring the decrease in absorbance at 340 nm (UVA) as NADH is oxidized to NAD+.
• Normal RR for AST < 35 U/L (IU/L), but can vary by laboratory.
• ALT needs a coupled with the Lactase Dehydrogenase

GGT

• The Gamma glutamyltransferase help regulate and maintain extracellular reduced glutathione(GSH)
• The is a major antioxident in tri-peptide, and is made from Glycine-Cysteine (N-acetylated)-Glutamine.
• GGT enzyme often uses glutathione.
• It primarily detects hepatobiliary disease with cholestasis, or biliary obstruction, associated with the highest elevation, It differentiates liver disease from bone disease:
• disorder/ALP/GGT:
• bone/elevated/normal
• liver/elevated/elevated

Alkaline phosphate (ALP)

• It is an enzyme that areas of concentrated in the body, functions and Multiple contains isoenzymes.
• Clinical significance:
• (Bile ducts of liver) of a hepatobiliary disease
• A bone disease
• Best understood functions:
• Bile acid production is in the liver
• osteoblast synthesis and growth in the bone
• Facilitates lipid transport in the intestine
• Multiple forms of ALP are in healthy human serum (liver and bone serum); Ref. ranges dependent on age and gender, good bone marker in early ages.
• Major clinical significance:
• Liver: thought to aid in bile production
• ALP frees inorganic and inorganic phosphate results from an organic phosphate monoester that is resulting the production alcohol to the pH 10 levels.
• elevated levels are important but necessary in bone and normal growth, and elevated levels are seen in adolescence
• The 4-nitrophenol-phosphate (4-NPP) is the substrate that creates yellow product
• 4-nitrophenoxide: measured at 405 nm (visible range), at pH 10.2.

Pancreatic Digestive Functions

• The pancreas is the source of amylase, lipase and protease.
• It produces amylase, glucagon, insulin, somatostatin, and pancreatic. Digestive system.
• Hormones or enzyme
• Amylase is a digestive (amylase) to start the digestive Process
• The amylase clinical significance is that it primarily uses Pancreatitis
• It's a hydralose and breaks carbs, including that starts starch, amylopectin, glycogen from it is partially broken items
• The region that is known to come from intestinal amylase (pancreas); majorly a clinical interest
• Lipase's work is that it helps hydrolyzes glycerol esters and fatty acids to allow the body to produce monoglyceride
• Serum lipase elevates if obstructed
• The half life of pancreatic amylase is longer than a normal amylase
• Expected values: = < 38 U/L.

Hematology/Oncology

• LD enzyme transfers a hydrogen to catalyzation and oxidation of L-Tactate
• Platelets and RBC rich LD sources.
• Function is in aerobic environment
• ISO is an enzymatic activity to is usually what is rare to measure.

Red and White Blood tests

• Hemolysis happens in anemic environments, where folate or the B12 absorption issue, also leukemia are present.
• Platelets are rich in LD.
• serum sample and reject hemolized samples

Acid Phosphatase (ACP)

• Hyradolase with water to break bonds
• Causes is values in the clinical test increases like with
• --prostate cancer
• --Some disorder broken bone
• --Gaucher breaks-bone issues but it may be good to diagnose