| dc.description.abstract | The kinetic mechanism of rat liver hexokinase D (‘ glucokinase’)
was studied under non-co-operative conditions with 2-
deoxyglucose as substrate, chosen to avoid uncertainties derived
from the co-operativity observed with the physiological substrate,
glucose. The enzyme shows hyperbolic kinetics with respect to
both 2-deoxyglucose and MgATP#−, and the reaction follows
a ternary-complex mechanism with Km¯19.2³2.3 mM for 2-
deoxyglucose and 0.56³0.05 mM for MgATP#−. Product inhibition by MgADP− was mixed with respect to MgATP#− and
was largely competitive with respect to 2-deoxyglucose,
suggesting an ordered mechanism with 2-deoxyglucose as first
substrate and MgADP− as last product. Dead-end inhibition by
N-acetylglucosamine, AMP and the inert complex CrATP [the
complex of ATP with chromium in the 3 oxidation state, i.e.
Cr(III)–ATP], studied with respect to both substrates, also
supports an ordered mechanism with 2-deoxyglucose as first
substrate. AMP appears to bind both to the free enzyme and to
INTRODUCTION
Hexokinase D (ATP:-glucose 6-phosphotransferase, EC
2.7.1.1), often known as glucokinase or hexokinase IV, is one of
the four glucose-phosphorylating isoenzymes present in vertebrates and plays the role of a glucose sensor in pancreatic β-cells
and in hepatocytes [1,2]. Mutations in its gene in humans are
correlated with a type of diabetes, namely maturity onset diabetes
of the young (‘MODY’) [3]. The enzyme shows a sigmoidal
dependence of rate on the glucose concentration [4–6], a property
of kinetic origin that makes the enzyme sensitive to changes in
the blood glucose level.
Although the main models for explaining the cooperative
behaviour of hexokinase D have postulated an ordered addition
of substrates, with glucose as first substrate [7–10], the subject is
not closed, and it remains unclear whether the kinetic mechanism
is totally ordered. Isotope-exchange studies at equilibrium
suggested a small degree of randomness, but no binary
EE
MgATP#− complex could be detected [11]. The order of product
release has not been clearly established either, on account of the
co-operative behaviour, which complicates the data analysis.
Some kinetic studies [7] suggested MgADP− as the last product,
Abbreviations used: CrATP, the complex of ATP with chromium in the 3 oxidation state, i.e. Cr(III)–ATP; dGlc (when used in the name of a complex
or as part of a kinetic symbol), 2-deoxyglucose; GlcNAc (when used in the name of a complex), N-acetylglucosamine; Glc6P (when used in the name
of a complex), glucose 6-phosphate; Nbs2, 5,5«-dithiobis-(2-nitrobenzoic acid). 1 This paper is dedicated to the memory of Professor Hermann Niemeyer, whose influence on hexokinase research remains strong. 2 To whom correspondence should be addressed (e-mail cardenas!ibsm.cnrs-mrs.fr).
the EE
dGlc complex. Experiments involving protection against
inactivation by 5,5«-dithiobis-(2-nitrobenzoic acid) support the
existence of the EE
MgADP− and EE
AMP complexes suggested by
the kinetic studies. MgADP−, AMP, 2-deoxyglucose, glucose
and mannose were strong protectors, supporting the existence of
binary complexes with the enzyme. Glucose 6-phosphate failed
to protect, even at concentrations as high as 100 mM, and
MgATP#− protected only slightly (12%). The inactivation results
support the postulated ordered mechanism with 2-deoxyglucose
as first substrate and MgADP− as last product. In addition, the
straight-line dependence observed when the reciprocal value of the inactivation constant was plotted against the sugar-ligand concentration supports the view that there is just one sugarbinding site in hexokinase D. | |
