Insight into the biochemical characteristics of a novel glucokinase gene mutation

Abstract

Glucokinase (GCK) acts as a glucose sensor and regulates β-cell insulin secretion. The heterozygous mutations in the gene encoding GCK cause a reduction of the enzyme activity, which results in a monogenic form of diabetes, maturity-onset diabetes of the young. In the present study, we identified and functionally characterized a novel missense mutation in the GCK gene, which results in a protein mutation Glu(339)→Lys (E339K), from a Chinese family with hyperglycemia. The same GCK mutation that co-segregated with diabetes phenotype was identified in five members of this family but was not found in 200 healthy control individuals. We expressed and affinity-purified the GCK proteins from bacterial expression system that carries mutation (E339K) and fused to glutathione S-transferase. The expressed GCK protein was subjected to the measurement of its biochemical effects of the missense mutation on GCK activity. Our results showed that the mutation reduced the GCK protein yield. The enzymatic kinetics and the thermal stability analysis on the recombinant GCK proteins revealed that the mutation inactivates enzyme kinetics and severely impaired the GCK protein stability.

Phenotypic severity of homozygous GCK mutations causing neonatal or childhood-onset diabetes is primarily mediated through effects on protein stability

“…This has been previously proposed for some naturally occurring GCK-MODY mutations (10–19,21,31), including a few that have also been studied in mice (20,32), but has never been systematically investigated in such a large group of patients with homozygous GCK mutations. Furthermore, our results indicate that protein stability may be the principal determinant of phenotypic severity for all but the most severely kinetically defective mutations.…”

Crystal structure of E339K mutated human glucokinase reveals changes in the ATP binding site

“…A novel mutation, E339K, was found in a Chinese family by Shen et al [13]. According to clinical features and laboratory examinations, the patients were diagnosed with fasting hyperglycemia.…”

“…According to clinical features and laboratory examinations, the patients were diagnosed with fasting hyperglycemia. The kinetics analysis showed lower affinity for glucose and ATP, resulting in the dramatically decreased relative activity of E339K [13]. To better understand the mechanism of how E339K mutation is associated with hyperglycemic phenotype, we determined the first GK mutant crystal structure and showed its structural differences with wild-type GK.…”

“…Their structure provides a general explanation of how the enzyme activity of GK can be affected by some of the mutations, especially those located in the glucose and ATP binding pockets and those involved in the conformation transition. Recently, a new inactivating mutation E339K was found by Shen et al in a Chinese family with hyperglycemia [13]. This E339K mutation, however, is situated far from the ATP binding site.…”

Heterozygous lys169Glu mutation of glucokinase gene in a Chinese family having glucokinase-maturity-onset diabetes of the young (GCK-MODY)

“…Direct sequencing of the entire coding region and intron-exon boundaries of the GCK gene (RefSeq NM_000162.3) was performed as described before[3] in this family and 200 healthy controls. A heterozygous missense mutation, i.e., NM_001354800.1:c.505A>G (p. Lys169Glu) as the Human Genome Variation Society name of this variant [Figure 1b], was identified.…”