Date of Award
Master of Science
Biochemistry, Biophysics and Molecular Biology
The binding of hexokinase Types I and II to mitochondria is a putative checkpoint in apoptosis. Evidence supports the specific binding of residues 1 through 15 of hexokinase Type I (HKI) to the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane. Ala4 and Ala8 of HKI play critical roles in mitochondrial binding. Substitution of these residues individually with leucine causes the loss of binding properties. Identified here is the significant role of the methionine at position 1 in the binding of HKI to mitochondria. The following conclusions can be drawn from the properties of mutant HKI constructs: Recombinant wild-type HKI is blocked at the N-terminus. The replacement of Ile2 with threonine results in a recombinant HKI with an unblocked amino terminus that does not bind to mitochondria. The extension of HKI at the N-terminus by duplicating its first four residues (putting amino acids at positions 0, -1, -2, and -3) retains mitochondrial binding properties. The mutation of Ile at position -2 to threonine results in an unblocked amino terminus that still binds to mitochondria. The mutation of Met1 to leucine in the extended construct greatly reduces mitochondrial binding. The conclusion: blocking of the N-terminal methionine is not a significant factor in mitochondrial binding of HKI. Instead, it is the specific structure of the methionine side chain that enables a strong binding interaction. Side chains at positions 1, 4, and 8 define a contiguous face on the N-terminal α-helix of HKI critical mitochondrial binding.
Maskey, Manjit, "Role of the N-terminus of Recombinant Human Brain Hexokinase in Mitochondrial Binding" (2012). Graduate Theses and Dissertations. 12864.