lecture 18 enzymes Flashcards
role of enzymes in biochemical rxn
start or speed up rxn of conversion of reactants to products
relationship btw substrate and enzyme active site
active site of enzyme binds to substrate to undergo chemical run
3 ways progress of enzyme catalyzed rxn is monitored
disappearance of substrate, appearance of product, conversion of cofactor
what is happening in lag phase
run rate is zero but then substrate start binding to active site, rxn rate incr. once [ES] constant measurements taken
what is happening in linear phase
all active sites occupied. [ES] constant. depends only on [enzyme]. independent of [substrate] zero order kinetics
what is happening in plateau phase
rxn rate declines as substrate consumed, becomes dependent on [substrate], and becomes non linear. first-order kinetic
diff btw cont and fixed time measurement of an enzyme analyte
cont- multiple point measurement
fixed- single point, must know time over which ran is linear
why fixed-time measurement enzyme assay only be performed once rate of enzyme reaction is known?
discontinuous, needs to be in the linear phase to be valid
why hexokinase method for glucose measurement called coupled enzyme rxn
the first enzyme rxn produces the substrate for the second enzyme rxn
compare enzyme or substrate is rate limiting
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compare measuring serum enzyme using reagent substrate vs measuring serum substrate using enzyme reagent
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what is EcoRI DNA restriction digest
cuts each DNA strand by hydrolyzing phosphodiester linkage betn each G-A, leaving two ‘sticky-ends’ that are complementary
what is EcoRI DNA restriction digest
joins dsDNA end, phophosphodiester linkages can reform, recreating the EcoRI restriction site
what is DNA ligase
- Taq DNA polymerase
- DNA sample to be tested
- Reaction buffer
- MgCl2
- dNTP (deoxynucleotides, G, A, T, and C)
- Oligonucleotide primers
what are components of PCR amplication
