Lab 1 Flashcards
NADPH is essential for
anabolic reactions in metabolism (building)
NADPH is produced by the
pentose phosphate pathway
also provides ribose-5-phosphate for nucleic acid biosynthesis.
The first enzyme on the pentose phosphate pathway is
glucose-6-phosphate dehydrogenase
glucose-6-phosphate dehydrogenase
catalyses the oxidation of
G6P to 6-phosphogluconate
with the concomitant reduction of NADP+ to give NADPH.
the reaction is irreversible and highly regulated by the availability of the NADP+ cofactor.
Glucose-6-phosphate dehydrogenase is strongly inhibited by
NADPH and by fatty acid esters of coenzyme A.
Chemically this reaction is the oxidation of
RCHO to RCOO-
The phosphate pentose pathway is carried out in
the cytosol
predominantly in the liver, which is also the major site of gluconeogenesis.
The two pathways are directly linked by glucose-6-phosphate, a key intermediate on both pathways
Isomerisation of fructose-6-phosphate by glucose-6-phosphate isomerase provides
glucose-6-phosphate G6P
Glucose-6-phosphate isomerase also acts as a
cytokine, which stimulates cell motility and is associated with tumour development and metastasis
Since the reaction catalysed by glucose-6-phosphate isomerase does not produce a direct change in UV absorption, we will be coupling it
with glucose-6-phosphate dehydrogenase and measuring the rate of NADPH production at 340 nm.
In the class, we will be assaying the enzyme glucose-6-phosphate isomerase. Why are we coupling this reaction glucose-6-phosphate dehydrogenase?
Because the reaction does not produce a direct change in UV absorption
In the experiment, what are we measuring at 340 nm?
The production of NADPH by glucose-6-phosphate dehydrogenase
Why do we add magnesium acetate to the enzyme assay?
Mg2+ is required by glucose-6-phosphate isomerase for activity
Why is the Tris.HCl added to the reaction?
It increases membrane permeability of cell membranes
It acts as a buffer for the reaction
In this experiment we will NOT add both enzymes at the same time to the reaction mixture. This is because:
Any contaminating glucose-6-phosphate in the fructose-6-phosphate needs to be removed first
In this experiment we are using a coupled assay because:
The product of glucose-6-phosphate isomerase is difficult to measure, so we couple to provide an easily measured product
When setting up a coupled assay, which of these features would we not wish to see?
The coupling enzyme works slower than the study enzyme in the quantities available
What is an optimal range of substrates for measuring Km?
Km and three two-fold dilutions above and below
summary
in the lab we want to find Km for G6P isomerase, which produces G6P as a product. we are coupling it to G6P dehydrogenase which uses G6P as a substrate to produce 6 phosphogluconate in the pentose phosphate pathway. this reaction also uses NADP+ to produce NADPH. The levels of NADPH is what is measured at 340nm.
