Question

1. One of the roles of the liver is to maintain a blood glucose level between 70 – 120 mg/dL (roughly 3.8 – 6.7 mM). Several factors are involved in the fact that liver cells (hepatocytes) respond differently than other cell types; on of those factors is differential expression of hexokinase isoforms. Most cells express hexokinase I; the liver primarily uses isoform IV, more commonly referred to as glucokinase. The table below summarized some of the differences between these isoforms.

Trait	Hexokinase	Glucokinase
KM	~0.1 mM	~10 mM
Relative vmax	1	0.75
Shape of kinetics curve with only glucose present, low ATP concentration	Hyperbolic	Sigmoidal
Inhibited by glucose-6-phosphate	Yes	No

a. Define the term isoform. How can isoforms of the same enzyme be differentially regulated?

b. Explain the physiological logic behind the differences between these isoforms.

c. Using the data provided, sketch a plot of enzyme rate as a function of glucose concentration for both isoforms on a single set of axes. Include curves for each isoform in the presence and absence of glucose-6-phosphate. Explain the reasoning behind your curves, including any decision as the type of inhibition exhibited by glucose-6-phosphate.

d. Hexokinase is also inhibited by ATP. Explain why this makes sense and draw a curve for hexokinase in the presence of ATP on the graph above.

Answer 1

a. Isoform are two or more proteins which are funtionally similar but not identical amino acid sequence. They can be encoded by different genes or by RNA transcripts from the same gene. This gene might have had different exons.

Human liver acyl-CoA oxidase isoforms 1a and 1b encoded by a single gene is a rate limiting enzyme in peroxisomal fatty acids beta-oxidation. Its deficiency could become lethal, or it may form a disease called pseudoneonatal-adrenoleukodystrophy. In this enzyme there are two mRNA variants which are transcribed from a single gene encode ACOX1a or ACOX1b isoforms. A mutation in spice site has been found in metabolic screening of a patient with fatal peroxisomal beta-oxidation defect. This resulted to defective peroxisomal fatty acids -oxidation. So, it has been shown in human liver these mRNA variants are expressed differentially.

d. The enzyme which catalyzes the phosphoryl group transfer is called Hexokinase. When it binds with glucose, it undergoes a conformatinal change and prevents the hydrolysis of ATP. Hexokinase phosphorylates hexoses (6 carbon sugars), forming hexose phosphate. Glucose-6-phosphate is the most important product of hexokinases. The most important sustrate is glucose. In Glycolysis, the enzyme Hexokinase uses ATP to transfer a phosphate to glucose to form glucose-6-phosphate. The gamma-phosphoryl group of an ATP molecule is transferred to the oxygen at the C-6 of glucose. In order to start this reaction, ATP forms a complex with magnesium 2 ion and glucose binds to exokinase. Hexokinase is inhibited by its product. Then, the level of glucose 6-phosphate rises because it is in equilibrium with fructose 6-phosphate. Therefore, the inhibition of phosphofructokinase leads to the inhibition of hexokinase.