Effect of ligand-induced conformational changes on the reactivity of specific sulfhydryl residues in rat brain hexokinase

Hutny, Jan; Wilson, John E.

doi:10.1016/0003-9861(90)90628-c

Cited by 17 publications

(1 citation statement)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The main problem that should now be solved concerns the previously reported evidence of a single high-affinity glucose 6phosphate-binding site for each 100 kDa enzyme molecule [7,8,37]. Some authors [22,38,39] have suggested that, although each mammalian hexokinase type I molecule contains two glucose 6-phosphate regulatory sites, only one of these (namely that localized in the N-terminal half) would be functioning in the intact enzyme, whereas the other would be latent. Although this hypothesis is fascinating, we cannot exclude the possibility that only the regulatory site in the catalytic domain could be implicated in hexokinase regulation.…”

Section: Discussionmentioning

confidence: 99%

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

Magnani

Bianchi

Casabianca

et al. 1992

Biochemical Journal

View full text Add to dashboard Cite

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.

show abstract