Enzymes Flashcards
Enzymes
proteins that act as biological catalysts
decrease delta G to facilitate reaction
normally inside the cells; only in blood when cells are damaged
Enzyme reaction factors
time substrate level pH of mixture temperature enzyme concentration activators/inhibitors that may be present
Inhibitors
competitive- binds to enzyme active site
uncompetitive- binds to ES complex
non-competitive - binds to other site & causes conformational change
reversible : can ‘dilute’ off
non reversible : alters active site permanently
Time
fixed or kinetic (series of times)
product formation different depending on the time
substrate
goal is to make so much substrate available so that it does not become a limiting factor & makes all the reactions a zero order reaction time
pH of mixture
most rx use pH of 7-8
temperature
optimume 37 in US; 30 in europe
enzyme concentration
originate from patient samples
activators/inhibitors
activators aid enzyme reaction to be optimum
inhibitors compete & reaction is not optimum (drugs in patient’s system etc)
Km
substrate concentration at half of Vmax
exceeding Km will give you a zero order reaction
Enzyme class names
oxidoreductases - lactic dehydrogenase transferases hydrolases lyases isomerases ligases
International Unit (IU)
the amount of enzyme which can convert 1 umol of substrate per minute to product
Systemic Unit (SI)
katal unit - amount of enzyme that catalyzes 1 mole of substrate per second
Isoenzymes
forms of an enzyme that are molecularly different but catalyze the same reaction
can usually be separated by electrophoresis or Ab-Ag reaction
Creatine Kinase
associated with ATP regeneration in muscle & transport systems
found in heart, muscle, & brain tissue
elevated CK associated with AMI, muscular dystrophy, CNS seizures etc etc etc
CK analyzer forward reaction
forward reaction
creatine + ATP w/ patient’s CK -> CrP + ADP
ADP + PEP –(PEP phophatase) –> ATP + pyruvate
pyruvate + NADH +H –(LD)-> lactate & NAD
NAD IS READ AT 340 nm
CK analyzer reverse rx
CrP + ADP --(CK)--> creatine + ATP ATP + glucose --(HK)--> G-6-P + ADP G-6-P + NADP --(G6PD)-> bleh + NADPH more commonly used CK is from patient sample! doesn't take as much activation energy for this reaction & works better on most machines
CK isoenzymes
M & B units in dimers - 3 isoforms
CK1 (BB)- brain tissue ; not usually found in serum
CK2 (MB) - heart muscle, increases 4-8 hr after AMI then normal after 48-72 hrs
CK3 (MM) - skeletal muscle, increases in muscle diseases
Aspartate Aminotransferase (AST)
transfers amino acid groups to make a new amino acid
coenzyme pyrioxal phosphate (vit B6)
found in cardiac, liver & skeletal tissue
SIGNIFICANT in viral hepatitis
AST analyzer methods
uses amino acid transferase reaction
substrate: L-aspartate
enzymes used: AST (patient’s) & malate dehydrogenase
reads NAD @ 340 nm
LD analyzer method
uses pyruvate to lactate & the change in the coenzyme NAD
pyruvate + NADH + H -(LD)–> lactate + NAD
reaction read at 340 nm
enzyme found only in the CYTOPLASM of cells
LD isoenzymes
uses H & M subunits in tetramers
heart attack see LD1> LD2 (flipped from normal)
LD4 & LD5 increase in liver issues
LD3 -pulmonary
after suspected heart attack what tests are usually ordered?
general lab tests to see if lungs, kidneys & systems are normal
specific tests: CK, AST, LD, & Troponin
tests ordered when liver involvement suspected
AST, LD, ALT, gamma -GT, ALP
Alanine Transferase (ALT)
found more in the liver than other tissues
viral hepatitis: ALT may increase 5-10x
liver cirrhosis: AST> ALT (usually less than 5x)
ALT analyzer method
amino transferase enzyme substrate: L-alanine ALT: patient enzyme 2nd enzyme: LD measure: NAD @ 340nm
Alkaline phosphatase (ALP)
NEEDS MG2+ as ACTIVATOR
found in most cell membranes as it is involved in the transfer of metabolites across cell membranes
ALP in serum: bone & liver
ALP associations
^^ ALP associated w/ liver or bone
liver biliary duct obstructions can lead to ^ ALP
bone diseases ^ ALP : ricketts, bone cancer, padgetts
hepatitis: modest ^ ALP
ALP method
ALP is a hydrolase enzyme
substrate:para-nitro-phenol phosphate (PNPP)
ALP: patient enyzme
turns yellow & read
ALP isoenzymes
5 possible isoenzymes
liver isoenzyme overlaps w/ bone isoenzyme
bone isoenzyme is not as heat stable as liver isoenzyme - heat stability isoenzyme separation technique
Gamma glutaryl transferase (Gamma-GT)
involved in the transfer of gamma-glutamyl group to other amino acid
found in all cells but muscle
recent alcohol consumption will falsely elevate GGT levels
not used very commonly
Amylase
found in salivary gland & pancreas
hydrolyzes a-1,4-glycosidic bonds ; functions to break up complex CHO
major increases in amylase in pancreatitis & mumps
Amylase methods
classic iodometric method
saccharogenic method
enzymatic methods: uses artificial maltose & measures NADH from patient amylase
Lipase
only pancreatic enzyme; breaks off fatty acids from triglycerides
stays elevated longer than amylase in pancreatitis
substrate: triolein
enzyme: patient lipase
measures dye color change
Amylase Reaction
substrate: maltose
patient enzyme: amylase
other enzymes: a-glucosidase, hexokinase, G6PD,
Read: NADH
