Functional organization of mammalian hexokinase II. Retention of catalytic and regulatory functions in both the NH2- and COOH-terminal halves.
The mammalian hexokinase (HK) family includes three closely related 100-kDa isoforms (HKI-III) that are thought to have arisen from a common 50-kDa precursor by gene duplication and tandem ligation. Previous studies of HKI indicated that a glucose 6-phosphate (Glu-6-P)-regulated catalytic site resides in the COOH-terminal half of the molecule and that the NH2-terminal half contains only a Glu-6-P binding site. In contrast, we now show that proteins representing both halves of human and rat HKII have catalytic activity and that each is inhibited by Glu-6-P. The intact enzyme and the NH2- and COOH-terminal halves of the enzyme each increase glucose utilization when expressed in Xenopus oocytes. Mutations corresponding to either Asp-209 or Asp-657 in the intact enzyme completely inactivate the NH2- and COOH-terminal half enzymes, respectively. Mutation of either of these sites results in a 50% reduction of activity in the 100-kDa enzyme. Mutation of both sites results in a complete loss of activity. This suggests that each half of the HKII molecule retains catalytic activity within the 100-kDa protein. These observations indicate that HKI and HKII are functionally distinct and have evolved differently.
Ardehali
H
H
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Yano
Y
Y
Printz
R L
RL
Koch
S
S
Whitesell
R R
RR
May
J M
JM
Granner
D K
DK
D000818:Animals; D001483:Base Sequence; D005075:Biological Evolution; D017931:DNA Primers / Q000737:chemistry; D005947:Glucose / Q000378:metabolism; D005958:Glucosephosphates / Q000378:metabolism; D006593:Hexokinase / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
D017931:DNA Primers; D005958:Glucosephosphates; D011994:Recombinant Proteins; D006593:Hexokinase; D005947:Glucose