ENZYMOLOGY
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*

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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 Arg801between 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 andKm 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 μm) 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.

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*

This research was supported in part by Grant NS 10546 from the National Institutes of Health, United States Public Health Service, Grant MCB-9603595 from the National Science Foundation, and Grant 95–37500-1926 from the United States Department of Agriculture. This is Journal Paper J17891 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, Project 3191, and supported by the Hatch Act and State of Iowa funds.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Biological Sciences, Carnegie Mellon University, 4400 Fifth Ave., Pittsburgh, PA, 15213.