Characterization of glucokinase mutations associated with maturity-onset diabetes of the young type 2 (MODY-2) - Different glucokinase defects lead to a common phenotype

Glucokinase (GK) is expressed in the pancreatic beta-cells and liver, and p lays a key role in the regulation of glucose homeostasis. The enzymatic act ivity and thermal stability of wild-type (WT) Oh and several mutant forms a ssociated with maturity-onset diabetes of the young type 2 (MODY-S) were de termined by a steady-state kinetic analysis of the purified expressed prote ins. The eight MODY-S mutations studied were Ala53Ser, Val367Met, Gly80Ala, Thr168Pro, Arg36Trp, Thr209Met, Cys213Arg, and Val226Met. These missense m utations were shown to have variable effects on GK kinetic activity. The Gl y80Ala and Thr168Pro mutations resulted in a large decrease in V-max and a complete loss of the cooperative behavior associated with glucose binding. In addition, the Gly80Ala mutation resulted in a sixfold increase in the ha lf-saturating substrate concentration (S-0.5) for ATP, and Thr168Pro result ed in eight- and sixfold increases in the S-0.5 values for ATP and glucose, respectively. The Thr209Met and Val226Met mutations exhibited three- and f ivefold increases, respectively, in the S0.5 for ATP, whereas the Cys213Arg mutation resulted in a fivefold increase in the S-0.5 for glucose. These m utations also led to a small yet significant reduction in V-max. Of all the mutations studied, only the Cys213Arg mutation had reduced enzymatic activ ity and decreased thermal stability. Two mutants, Ala53Ser and Val367Met, s howed kinetic and thermal stability properties similar to those of WT. Thes e mutants had increased sensitivities to the known negative effecters of GK activity, palmitoyl-CoA, and GK regulatory protein. Taken together, these results illustrate that the MODY-2 phenotype may be linked not only to kine tic alterations but also to the regulation of Oh activity.