Identification and analysis of novel R308K mutation in glucokinase of type 2 diabetic patient and its kinetic correlation

Abstract

Glucokinase (GK) plays a critical role in glucose homeostasis and the mutations in GK gene result in pathogenic complications known as Maturity Onset Diabetes of the Young 2, an autosomal dominant form of diabetic condition. In the present study, GK was purified from human liver tissue and the pure enzyme showed single band in SDS-PAGE with a molecular weight of 50 kDa. The kinetics of pure GK showed enzyme activity of 0.423±0.02 µM glucose-6-phosphate (G6P)/mL/Min and Km value of 6.66±0.02 µM. These values were compared in the liver biopsy of a clinically proven type 2 diabetic patient, where GK kinetics showed decreased enzyme activity of 0.16±0.025 µM G6P/mL/Min and increased Km of 23±0.9 µM, indicating the hyperglycemic condition in the patient. The genetic analysis of 10th exon of GK gene from this patient showed a R308K mutation. To substantiate these results, comparative molecular dynamics and docking studies were carried out where a higher docking score (-10.218 kcal/mol) was observed in the mutated GK than wild-type GK structure (-12.593 kcal/mol) indicating affinity variations for glucose. During the simulation process, glucose was expelled out from the mutant conformation but not from wild-type GK, making glucose unavailable for phosphorylation. Therefore, these results conclusively explain hyperglycemic condition in this patient.

Novel mutations in the kinase domain of BCR-ABL gene causing imatinib resistance in chronic myeloid leukemia patients

“…The ABL structure was retrieved from the Protein Data Bank (PDB ID: 1OPL) (http://www.rcsb.org/pdb/explore/explore.do?structureId=1OPL) with the resolution of 3.42 Å and was loaded into the MOE working environment ignoring all water molecules and heteroatoms. The structure was subjected to protonation followed by energy minimization under MMFF94x force field (Merck Molecular Force Field) 36,37 to an RMSD of 0.05 where the implicit solvated environment was specified and the stabilized conformation was saved in PDB format. The mutations identified in the ABL gene were introduced individually in the wild-type energy minimized ABL protein, the resultant structures was again subjected to energy minimization.…”

“…The mutations identified in the ABL gene were introduced individually in the wild-type energy minimized ABL protein, the resultant structures was again subjected to energy minimization. The energy minimization was done with the same parameter set and the stabilized conformation of the mutated ABL structures was saved individually in PDB file 36,37 .…”

“…The stabilized conformations of both wild-type and mutated ABL structures were subjected to molecular dynamics simulations 36,38 individually. To bring the system to perfect density or equilibration, 20 picoseconds (ps) and MMFF94x force field were applied for the MD simulations.…”

“…The initial temperature was set to 30 K and increased to a runtime temperature of 300 K with a heat time of 10 ps and the pH of the system was adjusted to 7.0. The constraints were applied to the light bonds of the protein and the constraints on water molecules were set to be rigid 36,38 .…”

“…The runtime temperature of 300 K and pressure of 100 kPa were set. NPA (Nose-Poincare-Anderson) algorithm was specified to solve the equations of motion, which was the most accurate and most sensitive method to generate true ensemble trajectories 36,38 . A constant temperature was maintained during the simulations with a bath relaxation time of 0.1 ps.…”