Abstract:
Hexokinase (E.C. 2.7. 11) from Dioscorea alata was isolated and
purified 80 - fold using a combination of ammonium sulphate
precipitation, gel filtration and ion-exchange chromatography.
Electrophoresis of the purified enzyme showed a single sharp
protein band which migrated anodically, with a molecular weight of
136,000 daltons as determined using gel filtration.
Initial velocity studies revealed that the enzyme operates via a
sequential mechanism with a compulsory formation of a ternary
complex of the form enzyme-glucose - MgA TpZ·. The Vmax for the
enzyme was determined to be 1.57 Umoles per minute. The Km for
glucose and MgATpZ' were found to be 3.2 x 10.1 and 8.0 x 10·4M
respectively, corresponding to the total free energy of binding of -
4.33 Kcal per mole. MgA TpZ' was found to inhibit the enzyme
competitively with respect to both glucose and MgA TpZ' in accord
with random order of substrate binding.
The dependence of the rate of reaction on temperature revealed a
break in the Arrhenius plot at 40°C with the activation energy of
11.13 kcallmole. Temperature stability studies indicated the enzyme
is considerably thermolabile with activation energy of thermal
denaturation of 31. 74 kcal per mole
