Enzymes Flashcards
Enzyme (mechanism of action)
- catalysts (not consumed in rxns)
- decrease activation energy needed to form product for more rapid formation
- S + E –> ES –> P + E
Factors affecting enzyme reactions
- time: fixed or kinetic
- substrate level
- pH of mixture (pH 7-8)
- temperature (30-37C)
- enzyme concentration
- activators/inhibitors
competitive inhibitors
bind to active site of enzyme
uncompetitive inhibitors
bind to ES complex
non-competitive inhibitors
bind to another site (not active site) on enzyme
Enzyme classes
- oxidoreductases
- transferases
- hydrolases
- lyases
- isomerases
- ligases
Enzyme unites of measure
- international unit (amt of enzyme which can convert 1 umole of substrate to product per min)
- systemic unit (amt of enzyme that catalyzes 1 mole of substrate per second = katal unit)
isoenzymes
- forms of enzymes that are only structurally different
- can catalyze same reaction as enzyme
- separated by electrophoresis or Ag-Ab rxn
Creatine kinase
- associated with ATP regeneration in muscle and transport systems
- found in heart, muscle, brain tissue
- elevations associated with AMI, muscular dystrophy, CNS seizures/shock, hypothyroidism, Reye’s syndrome, malignant hyperpyrexia
CK methods
- forward rxn: measure absorbance
- reverse rxn: commonly used method
CK isoenzymes
- M and B units of dimer
- CK1 or BB isoenzyme (brain)
- CK2 or MB isoenzyme (heart muscle)
- CK3 or MM isoenzyme (skeletal muscle)
Aspartate Aminotransferase (AST)
- transfers amino group to make new AA
- pyridoxal phosphate is coenzyme
- found in cardiac, liver and skeletal tissue
- significant in viral hepatitis
AST methods
- amino acid transferase rxn with alpha keto acid oxoglutarate
- read NAD absorbance
LD method
- uses pyruvate to lactate
- NAD absorbance is read
- increases of LD only if cell damage (because its a cytoplasmic enzyme)
LD isoenzymes
- uses H and M subunits in tetramers
- 5 possible isoenzymes
- LD1 > LD2 in heart attack
- LD4 and LD5 increase in liver disease
- LD3 is seen in pulmonary disorders
- LD6 is alcohol dehydrogenase
Lactate Dehydrogenase
- In AMI: rise, peak, remain elevated for 10 days
- in hepatic disorders: LD5 increase
Heart attack tests
CK, AST, LD and troponin
Liver disease tests
AST, LD, ALT, gamma-GT, ALP
Alanine Transferase (ALT)
- found in liver (liver specificity)
- increases in viral hepatitis, so does AST
- in cirrhosis AST > ALT
ALT method
- rxn is AA transfer from alaning to keto acid oxoglutarate
- read NAD absorbance
Alkaline Phosphatase (ALP)
- hydrolyzes phosphate groups from molecules at alkaline pH
- Mg is activator
- found in most cell membranes
- found in intestine, kidney, bone, liver and placenta
- increases associated with liver or bone
ALP method
- ALP is hydrolase enzyme
- method uses para-nitro-phenol phosphate (PNPP)
ALP isoenzymes
- 5 isoenzyme forms
- liver and bone overlaps on electrophoresis
- do immunochemical method for Bone ALP
- inhibition testing uses phenylalanine
Gamma-GT (Gamma glutaryl transferase)
- involved in the transfer of gamma-glutamyl group to other AA
- found in all cells but muscle
- originates in hepatobiliary system
- can increases with alcohol and cirrhosis
Amylase
- found in salivary glands and in pancreas
- 3 isoenzymes for salivary amylase, 3 for pancreatic amylase
- hydrolyzes alpha-1,4-glycosidic bonds
- increase seen in pancreatitis, mumps
Amylase methods
- Classic iodometric method
- Saccharogenic method
- Enzymatic method
- read absorbance
Classic iodometric method (amylase)
- iodine used to react with starch substrate
- when starch is all glucose, no iodine rxn
Saccharogenic method (amylase)
measured glucose formed by amylase action on starch substrate
Enzymatic method (amylase)
uses artificial substrate and measures glucose
Lipase
- pancreatic enzyme
- used in conjunction with amylase to identify pancreatitis
- method uses artificial substrate of triolein and links rxns to dye color change
