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Rat brain hexokinase: Glucose and glucose-6-phosphate binding sites and C-terminal amino acid of the purified enzyme

1974; Elsevier BV; Volume: 165; Issue: 2 Linguagem: Inglês

10.1016/0003-9861(74)90290-2

1096-0384

A C Chou, Jonathan E. Wilson,

Erythrocyte Function and Pathophysiology

The binding of glucose and glucose-6-P by pure rat brain hexokinase has been studied by using an ultrafiltration procedure [H. Paulus (1969) Anal. Biochem. 32, 91–100]. Each mole of enzyme (molecular weight 98,000) binds 1 mole of glucose or 1 mole of glucose-6-P. The dissociation constant for the enzyme-glucose complex (0.04 mm) is in excellent agreement with the kinetically determined Km for this substrate. The dissociation constant for the enzyme-glucose-6-P complex was estimated to be 1.3 μm, substantially lower than values of 7–8 μm obtained by alternative methods. This discrepancy appears to be due to retardation of the passage of the charged glucose-6-P through the ultrafiltration membrane, resulting in an effective increase in the ligand concentration at the membrane surface and thereby a decrease in the apparent dissociation constant. No appreciable retardation of the passage of the uncharged glucose molecule was observed. The binding of glucose-6-P (but not glucose) is prevented in the presence of Pi. This is in accord with a previously suggested model in which binding of Pi is considered to stabilize the enzyme in a conformation having little, if any, affinity for glucose-6-P. Serine was found as a C-terminal amino acid. The method used would not have detected C-terminal proline or tryptophan residues, and thus these cannot be excluded by the present experiments. However, in view of other results indicating that rat brain hexokinase consists of a single polypeptide chain, it seems probable that serine is indeed the only C-terminal amino acid in the molecule.