3a) Hexokinase/”glucokinase”

Once inside the cell the glucose is 6-phosphorylated by a hexokinase. There are four isoenzymes of hexokinase , hexokinase D, or type IV, is found only in liver and b-cell. This is the first control point for glycolysis since all cellular glucose transporters (i.e. excluding the intestinal brush border Na⁺-dependent one) are bi-directional and facilitate diffusion down a concentration gradient. Glucose 6-P is not transported thus the action of hexokinase traps the glucose in the cell. It can be released, however, if de-phosphorylated by glucose 6-phosphatase (only present in liver and kidney) the glucose can then escape into the blood.

“Mammals have several isoenzymes to catalyse the formation of G 6-P from glucose. Hexokinase IV, the predominant form in liver, is a low affinity hexokinase and differs significantly from the others. Its levels vary markedly with dietary and hormonal status; it requires much higher [glucose] (about 10mM) for half saturation, with a sigmoid dependence, and it is insensitive to physiological concentrations of G 6-P. It is thus well adapted to respond to variations in blood [glucose]. Despite its popular but misleading name of ‘glucokinase’ its sugar specificity is similar to that of other isoenzymes.” from “Text Book Errors: Hexokinase and ‘Glucokinase’ in liver metabolism” A. Cornish-Bowden and M.L. Cardenas, TIBS,16(1991)281.

ISOENZYME â	Hexokinase Type-I (A)	Hexokinase Type-II (B)	Hexokinase Type-III (C)	Hexokinase Type-IV (D)
È Characteristic				“Glucokinase”
Mol. Wt.	100 kDa	100 kDa	100 kDa	50 kDa
Km glucose	<1mM	<1mM	<1mM	~8mM*
Kinetic co-operativity with respect to glucose	no	no	no	*Yes (Hence not really a Km)
G 6-P inhibits allosterically	yes	yes	yes	no
Does Pi antagonize G 6-P inhib?	yes	no	?	-
G inhibits above ~0.5mM	no	no	yes	no
Subcellular location	Associated to porin on outer mito membrane	Also assoc. to mito?	Associated to nucleus	Nuclear & cytosolic
Inhibited by regulatory protein	no	no	no	yes

(From Cardenas, 1997, see below)

Glucokinase regulatory protein

In liver hexokinase IV only works at about 50% of its capacity, unless fructose is present, because of the existence of the glucokinase regulatory protein that binds to and acts as a competitive inhibitor of glucose for hexokinase IV. Now hexokinase IV does not normally convert fructose to F 6-P (which hexokinase I can) because of its very low affinity for fructose in the presence of glucose (they compete with each other for the enzyme and glucose usually wins), and liver has fructokinase which makes fructose 1-P. (F 1-P enters the glycolytic pathway in liver down by glyceraldehyde 3-P.) But F 6-P is made in liver from G 6-P by phosphoglucose isomerase which works so fast that the concentration of F 6-P is always in equilibrium in liver with that of G 6-P. At these physiological concentrations of F 6-P, it binds to the ‘glucokinase regulatory protein’ and together they inhibit hexokinase IV. Liver F 1-P from dietary fructose competes with F 6-P for the ‘glucokinase regulatory protein’, and when the ‘glucokinase regulatory protein’ has F 1-P bound to it, it does not inhibit hexokinase IV.

Hence liver phosphorylates glucose particularly quickly just after a meal containing fructose, but hardly at all at low blood glucose.

Hexokinase sigmoid kinetics.

An interesting detail that begs attention is that this single polypeptide chain enzyme is the only one discovered to date that exhibits sigmoid kinetics: all models put forward so far by enzymologists to explain sigmoid behaviour require co-operativity between the subunits of a multimeric protein.

Muscle, brain, adipose and other tissues have hexokinase I isoenzyme which is saturated, hence working ‘flat out’, even at low blood glucose levels, but this isoenzyme is inhibited directly by its own product G 6-P. So the rate of glucose phosphorylation in these other tissues does not rise very much after a meal.

Glucokinase gene mutation and diabetes.

It is also interesting to note that there is increasing evidence of a link between mutations in the glucokinase gene and MODY (maturity-onset diabetes of the young, “mild type-2” diabetes). Patients are heterozygous having one normal and one mutant allele, the latter showing reduced enzyme activity when expressed. Experimental homozygosity expressed in mice is lethal. Patients require a higher concentration of glucose to elicit insulin secretion compared to normal subjects.

Go to:

the next step in the glycolytic pathway – phosphoglucose isomerase

the carbohydrate metabolism contents page.

References:

1. An introduction to the isoenzymes of mammalian hexokinase types 1-111. J.E Wilson (1997) Biochem. Soc. Transac. 25, 103-107.

2. Regulation of overexpressed hexokinases in liver and islet cells. C.B. Newgard. (1997) Biochem. Soc. Transac. 25, 119-121.

3. Kinetic behaviour of vertebrate hexokinases with emphasis on hexokinase D (IV). M.L. Cardenas (1997) Biochem. Soc. Transac. 25, 131-135.

4. The regulatory protein of glucokinase. E. Van Schaftigen et al. (1997) Biochem. Soc. Transac. 25, 136-140.

5. Binding and translocation of glucokinase in hepatocytes. L. Agius (1997) Biochem. Soc. Transac. 25, 145-150.