A recombinant human ‘mini’-hexokinase is catalytically active and regulated by hexose 6-phosphates

Abstract

Mammalian hexokinase type I is a 100 kDa enzyme that has been considered to be evolved from an ancestral 50 kDa yeast-type hexokinase, insensitive to product inhibition, by gene duplication and fusion. According to this model, and based on many experimental data, the catalytic site is associated with the C-terminal half of the enzyme, although an allosteric site for the binding of glucose 6-phosphate could be present on the N-terminal half of the molecule. We have isolated a cDNA clone of hexokinase from a lambda gt11 human placenta library comprising 2658 bp, containing a single open reading frame of 1893 nucleotides, which encodes a truncate form of hexokinase starting from asparagine-287 to the terminal serine-917. This clone was further digested with restriction enzyme NcoI to obtain almost only the C-terminal half of human hexokinase starting from methionine-455 to the terminal amino acid and was overexpressed in active form in Escherichia coli and purified by ion-exchange h.p.l.c. The overexpressed 'mini'-hexokinase was found not only to catalyse glucose phosphorylation, but also to be inhibited by glucose 6-phosphate and other mono- and bis-phosphate sugars exactly like the complete mammalian enzyme. These results suggest that the C-terminal half of human hexokinase, in addition to the catalytic site, also contains the regulatory site and that the evolutionary relationship between the hexokinases should be reconsidered by including the appearance of a regulatory site before the gene duplication.

Glucose Phosphorylation and Mitochondrial Binding Are Required for the Protective Effects of Hexokinases I and II

“…The protein and gene structures of the 100-kDa enzymes suggest that they evolved from gene duplication and fusion of an ancestral 50-kDa enzyme. In HKI and HKIII, only their C-terminal halves retained catalytic activity (6,7,10,27,35,45,54,56), while both halves of HKII are catalytically active and sensitive to G6P (5). There is also functional interaction between the N-and C-terminal halves of HKII, as glucose binding by the N-terminal half causes the activity of the C-terminal half to be regulated at significantly lower concentrations of G6P (4).…”

Dual Mechanisms for Glucose 6-Phosphate Inhibition of Human Brain Hexokinase

“…The two halves (C-terminal and N-terminal) share significant sequence homology (24). Catalytic activity of the enzyme is associated with the C-terminal half of HKI (14,15,25,26), whereas the N-terminal half has a high affinity site for P i putatively responsible for the relief of G6P inhibition (14,15). Arora et al (14) have suggested that the binding of G6P to this site releases HKI from mitochondria and is not involved in inhibition.…”

“…Exactly how G6P functions as an inhibitor of HKI is unsettled (11,12). Although most investigators now believe that G6P competes with ATP at the active site of the enzyme (13)(14)(15)(16)(17), others suggest that G6P exerts its effect by binding to an allosteric site topologically distinct from the active site (12,18,19). On the other hand, there seems to be general agreement regarding the kinetic mechanism of HKI as being rapid-equilibrium Random Bi Bi (20 -22).…”

Identification of a Phosphate Regulatory Site and a Low Affinity Binding Site for Glucose 6-Phosphate in the N-terminal Half of Human Brain Hexokinase

“…On the other hand, the N-terminal half of hexokinase I is involved in the P i -induced relief of product inhibition on the basis of the following. (i) The isolated C-terminal half loses the property of P i -induced relief of Glu-6-P inhibition (13,16,17). (ii) A chimeric hexokinase containing the N-terminal half of hexokinase I and C-terminal half of hexokinase II exhibits P i -induced relief of Glu-6-P inhibition (18).…”