Enzymes and Protein Isoforms

Enzyme
proteins that are catalysts of biochemical reactions.
Substrate
reactant that binds to enzyme and is converted to product
Product
final molecule resulting from reaction
reaction rate
amount of product produced per unit of time
Active site
region of the enzyme where the substrate binds.
Apoenzyme
requires an additional non protein prosthetic group to function
cofactor
ions or smaller organic molecules needed for the activity of specific enzymes (non-protein)
Coenzyme
organic molecules derived from niacin, riboflavin and other vitamins. needed for activity of specific enzymes. (non-protein)
Holoenzyme
complete enyme-cofactor complex
Inhibitor
reduce catalytic activity of an enzyme
Zymogen
inactive enzyme precursor
Isoenzyme
structurally related protein products of different genes that catalyze the same reaction
Coupled Reaction
reactions that share a common intermediate
Enzyme Properties
Biological Catalysts
High Substrate Specificity
Lock and Key Theory
based upon the moleculare shape of the enzyme active site and substrate. If they match, reaction will occur
Induced Fit Model
enzyme reacts with the substrate causing changes in the configuration of the active site, allowing them to fit together and reaction to occur
Transfer (transferase)
catalyzes the transfer of groups, not including hydrogen
Redox (Oxidoreductase)
catalyze oxidation/reduction reactions
Hydrolytic (hydrolase)
catalyze hydrolysis reactions
Ligase
catalyzes the covalent bond of two molecules coupled with the hydrolysis ATP bonds
Isomerase
catalyzes the interconversion of structures or geotric changes in a molecule
Lyase
Catalyzes the formation of double bonds
Common Digestive Enzymes
Amylase, lipase, bile salts, peptidases, sodium bicarbonate
Amylase
digests startches to disaccharides
lipase
further digests dietary fats into fatty acids and gylcerol
bile salts
involved in the emulsification process of lipid absorbtion
Peptidases
break specific bonds (limited proteolysis) or break down complete peptide (unlimited proteolysis)
Pepsin
begins digesting of proteins by hydrolyzing them to smaller polypeptides
Trypsinogen
converted into trypsin. digests polypeptides into smaller amino acids
Sodium Bicarbonate
alkaline secretion that neurailizes tummy juice as it enters the small intestine.
Competitive Inhibitors
directly compete with the substrate for binding with the active site
noncompetitive inhibitors
weakly bind to an allosteric regulatory site on the enzyme. Do not compete with substrate.
Bilirubin
principle pigment in bile derived from the breakdown of hemoglobin
cirrhosis
abnormal structure and function of the liver. caused by liver disease.
excretory
discharge of waste from organ
secretory
process by which substances are released from an organ for a particular purpose
Hepatitis
inflammation of the liver
Jaundice
yellowish staining of the skin and sclerae, caused by high levels of bilirubin
NEcrosis
death of living cells or tissues
REticuloendothelial System (RES)
cels that can take up and sequester inert particles and vital dyes, includes macrophage and precursors.
Lobules
hexagonal plates of cuboidal hepatic cells, microscopin functional unit of the liver
Sinusoids
represent the main blood supply to the liver, located between the plates of hepatic cells.
Kupffer Cells
phagocytic cells, line the sinusoids, remove bacteria and other foreign particles from the blood.
Canaliculi
interlobular bile ducts,
Biliary Tract
network of ducts that connect the liver, gallbladder and duodenum
Hepatic Bile Duct
receives bile from the canaliculi, where bile exits the liver.
Cystic Ducts
ducts that deliver bile from the gallbladder
Common bile duct
delivers bile to the duodenum which aids in digestion
Portal Vein
delivers blood rich in digestive end products to the liver. 2/3 of the hepatic blood supply
Hepatic Artery
delivers oxygen rich blood to the liver. 1/2 of the livers blood supply
Hepatic Vein
blood exits the liver.
Functions of the liver
Excretory
Metabolism & Storage
Detoxification
Filtration
Excretory Function of the Liver
excretes bilirubin, cholesterol and drugs
Metabolism Function of the Liver
Liver metabolises and stores lipids, proteins, vitamins and minerals.
Dextoxification Function of the Liver
liver converts toxic and relatively insoluable compounds into less toxic compounds
Filtration Function of the Liver
the Kupffer cells remove bacteria and particles from the blood.
FActors that Influence REaction RAtes
Enzyme concentration, substrate concentration, preneces of conenzymes/cofactors, temperature, PH, inhibitors, organic solvents, salt concentration
Enzyme Concentration affects reaction rate…
increased products per time as enzymes increase
Substrate concentration affects reaction rate…
exymes works at max velocity when enzyme concentration is directly proportional to substrate concentration
Temperature affects reaction rate…
increasing temps usually increases reaction rate. Molecules move faster.
pH affects reaction rate…
other than optimal pH values, enzyme activity is affected because of structure alterations
ORganic Solvents
cause proteins to denture by disrupting hydrogen bonds
Allosteric Enzyme
has both an active site and an allosteric site to which effectors can bind. effectors binding causes the active site to change.
NEgative Allosterism
loss or reduction of product of the enzyme
Positive allosterism
additional product generated
Feedback Inhibition
frequently occurs in metabolic pathways. Excess final product binds to an earlier enzyme and inactivates it.
Protein Modification
Chemical groups are added or removed. can activate or inactivate the enzyme. Seen with phosphorylation
Testing Methodology for Aspartate Aminotransferase (AST)
Enzymatic Reaction. Spectrophotmetrically measured at 340 nm over time. Nad+ is directional proportional to AST
Specimen requirements for AST
serum, non hemolyzed, fasting, free of lipemia
Testing MEthod for Alanine Aminotransferase (ALT)
enzymatic reaction, spectrophotometricly measured at 340nm over time, to measure NADH which is directly proportional to ALT.
Specimen Requirements for ALT
Serum, fasting specimen, non hemolyzed, free of lipemia
Testing Method fro Gamma-glutamyltransferase (GGT)
Enzymatic Analysis. Gycerin is added, the product p-nitroaniline is measured at 405nm and 37C
Speciment requirements for GGT
Fasting speciment, non hemolyzed, free of lipemia, serum.
Testing Method for Alkaline Phosphatase (ALP)
Enzymatic analysis. The production of nitorphenoxide is spectrophotometrically measure at 405 nm.
Specimen Requirements for ALP
Serum, fasting, non hemolyzed, free of lipemia
All Testing Methods of Bilirubin
Modifed Evelyn-Malloy Method
Jendrassik and Grof Method
Direct Spectrophotmetry
Specimen requirements for Bilirubin
fasting, non hemolyzed, free of lipemia, protect from light.
Modified Evely-Malloy Method
Diazo reagent added to measure conjugated bilirubin at 540nm.
Methanol reagent added to develop unconjugated bilirubin, total protein thein measured at 540 nm.
very susceptible to hemolysis
Jendrassik and Grof MEthod
Most commonly used method. Sodium benzoate, followed by diazotized sulfanilic acid are incubate with the sample, then ascorbic acid, alkaline titrate, and dilute HCl are added, the resulting blue azobilirubin is measure at 600 nm.
Direct Spectrophotometry (Neonatal Bilirubin)
Limited to infants because of strong interference in older children and adults, measured at 454nm, then the easurement at 540nm is subtracted to account for Hb.
Function of AST
Catalyzes the transfer of an amino group or amino acid between aspartate and alpho-keto acids.
Sources of AST
Primary: Liver and heart
Secondary: skeletal muscle, kidney, pancreas
Clinical Significance of AST
widely used in identifying necrosis and inflammation of the liver. It elevates rapidly at the beginning of the disease.
Elevated EST is seen in:
Chronic Hepatitis
Cirrhotic Liver
Muscular dystrophry, dermatomyositis, and pulmonary emboli
Acute pancreatitis, crash injuries, gangrene, hemolytic disease
Function of ALT
catalyzes the reversile transfer of an amine group from the amino acids alanine and glutamate to alpha-ketoglutarate and pyruvate.
Sources of ALT
widely distributed through the body, liver is the main source, also concentrated in the kidney
Clinical Significance of ALT
more useful than AST in viral hepatitits and cholestatic disease, increases rapidly during hepatitis 15 to 20 times, used to screen blood donars for viral hepatitis.
Function of GGT
aids in the transport of amino acids through cell membranes, and involved in glutathione metabolism
Sources of GGT
liver and renal tubules highest activity, has been found in other places.
Clinical Significance of GGT
elevated in all liver diseases, highest concentration from biliary obstructions, primary or secondary liver cancers, extremely sensitive, non specific, used as a screentest for alcohol abuse.
Function of ALP
associated with bone calcification and lipid transport
Sources of ALP
Main source: Liver and Bone
Secondary: Spleen, Kidney, Intestines,
Clinical Significance of ALP
To detect hepatobiliary disease, liver responds by synthesis of ALP, Bone disease shows the highest levels.
Ammonia
toxic compound, normally formed in the body from the breakdown of proteins
Clinical Significance of NH3 Levels
dx of Reye’s syndrome, increases are caused by the liver’s inability to metabolize ammonia. can cause decreased mental capacity, stupor, coma, death
Bilirubin clinical significance
increase can cause jaundice
can also indicate Gilbert’s and Grigler-Najjar syndromes
Jaundice
yellow discoloration of the plasma, skin, and mucous membranes caused by high concentrations of bilirubin
Physiologic Jaundice
Most common hyperbilirubinemia, unconjugated common in neonate, levels greater than 18-20mg can cause brain damage, also caused by hemolytic anemias.
Hepatic Jaundice
conjugated and unconjugated bilirubin
usually due to a defect in bilirubin transport to the liver.
Posthepatic Jaundice
conjugated hyperbilirubinemia, impaired excretion, caused by obstruction of bile fulow to the intestines due to gallstones or tumor in the bile duct.
Cirrhosis
caused by excessive hepatic damage. irreversible scarring with formation of fibrotic connective tissue. Loss of functioning liver cells and impaired blood flow.
Hepatitis
inflammation of the liver caused by infections, radiation, drugs, chemicals or toxins
Hep A
transmitted by fecal-oral route by contaminated food or water.
Hep B
transmitted by blood, body fluids, and sexual routes.
HEpatic Tumors
cancers tumors from the liver, often associated with cirrhosis or Hep B
Ethanol
the most abused drug that affects the liver, leading cause of cirrhosis in the US
Acetiminophen
large doses produce fatal hepatic necrosis unles rapid treatment occurs