Hexokinase

I.U.B.: 2.7.1.1

Hexokinase catalyzes the reaction:

Hexokinases have been isolated from the yeast cell in two distinct forms, designated P-I and P-II (Schulze et al. 1969). These are separate, noninterconvertible isozymes (Womack et al. 1973).

Hexokinase is used to determine glucose, fructose, mannose and ATP.

Characteristics of Hexokinase from Yeast:

Molecular weight: The native forms have molecular weights of about 100,000 (Schulze et al. 1969) and consist of polypeptide chains of molecular weights slightly higher than 50,000 (Schmidt et al. 1973).

Optimum pH: 7.5 - 9.0 (Sols et al. 1958).

Composition: Both P-I and P-II contain the same amino terminus, valine, and the same carboxy terminus, alanine. Amino acid composition has been reported by Schmidt et al. (1973b).

Extinction coefficient: = 8.85 for P-I and 9.47 for P-II (Schmidt et al. 1973).

Isoelectric point: P-I, 5.25 and P-II, 4.93 (Schmidt et al. 1973).

Inhibitors: The enzyme is inhibited by compounds which react with SH groups. It is also inhibited by sorbose-1-phosphate, polyphosphates, 6-deoxy-6-fluoroglucose, 2-C-hydroxy-methylglucose, xylose and lyxose (Sols et al. 1958 and McDonald 1955).

Activators: Hexokinase requires magnesium ions for its catalytic activity. It is activated by catecholamines and related compounds (Harrison et al. 1972). Calcium ions do not affect the enzymatic activity.

Specificity: The enzyme phosphorylates D-fructose, 5-keto-D-fructose (Avigrad et al. 1968), D-glucose, 2-deoxy-D-glucose, D-mannose and D-glucosamine. ATP and ITP have been demonstrated to transphosphorylate in the yeast hexokinase reaction (Martinez 1961). The activity of P-I with fructose is 2.6 times that with glucose, whereas with P-II, a fructose:glucose ratio of 1:3 is obtained (Lazarus et al. 1966). The substrate specificities of yeast hexokinase have been extensively studied by Bessell et al. (1972).

Stability: Both the lyophilized preparation and the crystalline suspension are stable for 6-12 months at 2-8°C.

Assay

Method: The assay is based upon the reduction of NAD⁺ through a coupled reaction with glucose-6-phosphate dehydrogenase and is determined spectrophotometrically by measuring the increase in absorbance at 340 nm.

One unit of activity reduces one micromole of NAD⁺ per minute at 30°C and pH 8.0 under the specified conditions.

Reagents

• 0.05 M Tris*HCl buffer, pH 8.0 with 13.3 mM MgCl₂
• 0.67 M Glucose in above Tris⋅MgCl₂ buffer
• 16.5 mM Adenosine 5'Triphosphate in above Tris⋅MgCl₂ buffer
• 6.8 mM NAD in above Tris⋅MgCl₂ buffer

Note: NAD may vary in salt form and degree of hydration. Care should be exercised to use an analytical grade and the correct molecular weight.

Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (Worthington Code: ZF or ZFL). Dissolve at a concentration of 300 IU/ml in above Tris⋅MgCl₂ buffer. Store at 0 - 4°C during use.

Enzyme

Dissolve in Tris⋅MgCl₂ buffer, pH 8.0 to obtain a rate of 0.02 - 0.04 ΔA/min.

Procedure

Adjust spectrophotometer to 340 nm and 30°C.

Pipette into each cuvette as follows:

Tris⋅MgCl2 buffer	2.28 ml
0.67 M Glucose	0.50 ml
16.5 mM ATP	0.10 ml
6.8 mM NAD	0.10 ml
G-6-PDH	0.01 ml

Incubate in the spectrophotometer at 30°C for 6 - 8 minutes to achieve temperature equilibration and establish blank rate, if any. At zero time, add 0.1 ml of diluted hexokinase solution and mix thoroughly. Record increase in A₃₄₀ for 3-4 minutes. Determine ΔA/min from initial linear portion of curve.

Calculation