A study on the two binding sites of hexokinase on brain mitochondria

BACKGROUND: Type I hexokinase (HK-I) constitutes the predominant form of the enzyme in the brain, a major portion of which is associated with the outer mitochondrial membrane involving two sets of binding sites. In addition to the glucose-6-phosphate (G6P)-sensitive site (Type A), the enzyme is bound on a second set of sites (Type B) which are, while insensitive to G6P, totally releasable by use of high concentrations of chaotropic salts such as KSCN. Results obtained on release of HK-I from these "sites" suggested the possibility for the existence of distinct populations of the bound enzyme, differing in susceptibility to release by G6P. RESULTS: In the present study, the sensitivity of HK-I toward release by G6P (2 mM) and a low concentration of KSCN (45 mM) was investigated using rat brain, bovine brain and human brain mitochondria. Partial release from the G6P-insensitive site occurred without disruption of the mitochondrial membrane as a whole and as related to HK-I binding to the G6P-sensitive site. While, as expected, the sequential regime release-rebinding-release was observed on site A, no rebinding was detectable on site B, pre-treated with 45 mM KSCN. Also, no binding was detectable on mitochondria upon blocking site A for HK-I binding utilizing dicyclohexylcarbodiimide (DCCD), followed by subsequent treatment with KSCN. These observations while confirmed the previously-published results on the overall properties of the two sites, demonstrated for the first time that the reversible association of the enzyme on mitochondria is uniquely related to the Type A site. CONCLUSION: Use of very low concentrations of KSCN at about 10% of the level previously reported to cause total release of HK-I from the G6P- insensitive site, caused partial release from this site in a reproducible manner. In contrast to site A, no rebinding of the enzyme takes place on site B, suggesting that site A is 'the only physiologically-important site in relation to the release-rebinding of the enzyme which occur in response to the energy requirements of the brain. Based on the results presented, a possible physiological role for distribution of the enzyme between the two sites on the mitochondrion is proposed.