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.
Crystal structures of human hexokinase I reveal identical binding sites for phosphate and the 6-phosphoryl group of glucose 6-phosphate in proximity to Gly87, Ser88, Thr232, and Ser415, a binding site for the pyranose moiety of glucose 6-phosphate in proximity to Asp84, Asp413, and Ser449, and a single salt link involving Arg801 between the N- and C-terminal halves. Purified wild-type and mutant enzymes (Asp84 --> Ala, Gly87 --> Tyr, Ser88 --> Ala, Thr232 --> Ala, Asp413 --> Ala, Ser415 --> Ala, Ser449 --> Ala, and Arg801 --> Ala) were studied by kinetics and circular dichroism spectroscopy. All eight mutant hexokinases have kcat and Km values for substrates similar to those of wild-type hexokinase I. Inhibition of wild-type enzyme by 1,5-anhydroglucitol 6-phosphate is consistent with a high affinity binding site (Ki = 50 microM) and a second, low affinity binding site (Kii = 0.7 mM). The mutations of Asp84, Gly87, and Thr232 listed above eliminate inhibition because of the low affinity site, but none of the eight mutations influence Ki of the high affinity site. Relief of 1,5-anhydroglucitol 6-phosphate inhibition by phosphate for Asp84 --> Ala, Ser88 --> Ala, Ser415 --> Ala, Ser449 --> Ala and Arg801 --> Ala mutant enzymes is substantially less than that of wild-type hexokinase and completely absent in the Gly87 --> Tyr and Thr232 --> Ala mutants. The results support several conclusions. (i) The phosphate regulatory site is at the N-terminal domain as identified in crystal structures. (ii) The glucose 6-phosphate binding site at the N-terminal domain is a low affinity site and not the high affinity site associated with potent product inhibition. (iii) Arg801 participates in the regulatory mechanism of hexokinase I.
Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011, USA.
T Y
TY
Fang
O
O
Alechina
A E
AE
Aleshin
H J
HJ
Fromm
R B
RB
Honzatko
D001665:Binding Sites / Q000502:physiology; D001921:Brain / Q000201:enzymology*; D002942:Circular Dichroism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D006593:Hexokinase / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary
D004791:Enzyme Inhibitors; D010710:Phosphates; D019298:Glucose-6-Phosphate; D006593:Hexokinase