Two isozymes mediate the conversion of Glucose rightarrow Gl

Two isozymes mediate the conversion of Glucose rightarrow Glucose 6-P0_4. Hexokinase has a K_m of 23nM for Glucose, while Glucokinase has a K_m of 97nM for Glucose. If I had a mixture of the two enzymes, could I use an affinity column with Glucose as the ligand to separate the two proteins? If so, how? If not, why not? The enzyme \"E\" catalyzes the reaction A rightarrow B. The v_o for the reaction at a given [E], but different [A] is shown. Structural data shows that A can bind to E at two different sites. What is the MOST LIKELY explanation for all these data?

Solution

1. Hexokinase and Gluco kinases are Iso enzymes, both perform conversion of Glucose into Glucose-6-Phosphate. But their kinetics are different. Hexokinase located in all body cells and shows low Km and high affinity towards Glucose. That means even in lowest concentration of Glucose also it can bind and perform Reaction. Where as Glucokinase which is specifically located in the Liver, possess high Km and low affinity towards glucose. That means at highst concentrationof Glucose only it react with glucose and convert into Glucose-6-Phosphate. Based on the Data given above, Hexokinase is almost 4 times more affinity towards glucose than Glucokinase. So based on this data, affinity chromatography is used to seperate Gluco kinase and Hexokinase from mixture. To perform this Take affinity column with low concentration of glucose. Then pass the enzyme mixture through this column. Hexokinase, which has more affinity towards glucose, remain bind to thethe column and Glucokinase eluted out from the column, due to itssome lessPaul affinity. Later on Hexokinase also elutedin using solvents that distrub affinity interactions. In this way we can seperate the Glucokinase from hexokinase using affinity chromatography by using glucose as ligand