ADP-glucose pyrophosphorylase, an enzyme present as a homotetramer in Escherichia coli, plays a

vital regulatory role within the metabolic pathway of glycogen synthesis. Previous investigations into
the evolutionary divergence of this enzyme's role in plants have shown that the Lys42 residue present in
the catalytic domain is important in order for the enzyme to function. Therefore, the creation of
mutations by site-directed mutagenesis can lead to understanding of which characteristics make this
residue integral to the process of catalyzing reactions. By comparison of the mutant's kinetics with the
wildtype enzyme, we can begin to characterize this enzyme further.
Malachite Green assays provide a direct means of measuring the specific activity of our enzymes by
taking a product of the reaction pyrophosphate, cleaving it into single phosphates, and using a
spectrophotometer to make an absorbance reading. By creating curves with the enzyme's substrates
adenosine triphosphate and glucose 1-phosphate, as well as effector molecules such as magnesium and
fructose 1,6 bisphosphate, we can confirm conditions necessary for optimum catalysis and compare the
magnitude of difference in specific activities.
The creation of mutants in protein engineering is nothing new but it reveals important differences
within the functionality of an enzyme. By analyzing the data obtained, it can be shown which residues
increase or inhibit this metabolic pathway. This will shed light on the evolutionary distinctions between
various ADP-Glc Ppases and may prove important when working with plants to alter starch production
and produce more calorie-rich foods in order to meet an ever-demanding world population.