[The functional analysis of glucokinase gene E339K mutation].
OBJECTIVE
To explore the molecular mechanisms of glucokinase (GCK) E339K mutation resulting in maturity-onset diabetes of the young-2 (MODY2).


METHODS
Fasting plasma glucose (FPG), oral glucose tolerance test (OGTT) overload 2 h glucose (2hPG), glycosylated hemoglobin A1c (HbA1c) and fasting insulin (FIns) level were measured, respectively. Mutant glutathione S-transferase (GST)-GCK-cDNA was constructed with site-directed mutagenesis. Wild type and mutant GCK protein expressed in E. Coli were purified with affinity chromatography. Enzymatic kinetics and thermal stability were tested with enzyme-coupled analysis.


RESULTS
Compared with non-mutants, mutants had higher FPG [(6.92±0.95) mmol/L vs (4.70±0.35) mmol/L, P<0.001] 2hPG [(9.00±1.49) mmol/L vs (5.51±0.86) mmol/L, P<0.001], HbA1c [(6.46±0.69)% vs (4.83±0.30)%, P<0.01], and lower FIns level [(6.15±1.97) mIU/L vs (10.79±4.93) mIU/L, P<0.01], HOMA-ß (34.16±3.62 vs 172.53±76.58, P<0.001). This mutation induced lower protein yield [(12.7±1.72) mg/L vs (16.2±2.65) mg/L, P<0.01], lower appetency for glucose [S0.5: (13.96±1.89) mmol/L vs (5.92±0.99) mmol/L, P<0.001] and ATP [Km:(3.27±1.14) mmol/L vs (0.30±0.09) mmol/L, P<0.001], lower catalytic ability [Kcat: (1.62±0.35)/s vs (25.18±2.10)/s, P<0.001]. It also showed protein thermal instability.


CONCLUSIONS
Glucokinase gene E339K mutation promotes the development of MODY2 by affecting protein yield and protein stability as well as the enzymatic kinetics of GCK.
Shen
Yun-feng
YF
Department of Endocrinology, the Third Affiliated Hospital, Sun Yat-sen University, Diabetes Research Centre of Guangdong Province, Guangzhou 510630, China.
Liang
Hua
H
Cai
Meng-yin
MY
Weng
Jian-ping
JP
D044466:Asian People; D001786:Blood Glucose / Q000378:metabolism; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics* / Q000378:metabolism; D005941:Glucokinase / Q000235:genetics*; D005951:Glucose Tolerance Test; D006801:Humans; D009154:Mutation; D010375:Pedigree; D010957:Plasmids
D001786:Blood Glucose; D005941:Glucokinase