Identification and analysis of novel R308K mutation in glucokinase of type 2 diabetic patient and its kinetic correlation.
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.
Division of Animal Biotechnology, Department of Zoology, Sri Venkateswara University, Tirupati, Andhra Pradesh, India.
Nanda Kumar
NK
Yellapu
Koteswara Rao
KR
Valasani
Santhosh Kumar
SK
Pasupuleti
Sowjenya
S
Gopal
Sarma
S
Potukuchi Venkata Gurunadha Krishna
Bhaskar
B
Matcha
D000595:Amino Acid Sequence; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics*; D005941:Glucokinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D016415:Sequence Alignment
D005941:Glucokinase; D005947:Glucose
MODY2; enzyme activity; glucokinase; molecular docking; molecular dynamics