Glucokinase (GK) is expressed in the pancreatic β-cells and liver, and plays a key role in the regulation of glucose homeostasis. The enzymatic activity and thermal stability of wild-type (WT) GK and several mutant forms associated with maturity-onset diabetes of the young type 2 (MODY-2) were determined by a steady-state kinetic analysis of the purified expressed proteins. The eight MODY-2 mutations studied were Ala53Ser, Va1367Met, Gly80Ala, Thr168Pro, Arg36Trp, Thr209Met, Cys213Arg, and Va1226Met. These missense mutations were shown to have variable effects on GK kinetic activity. The Gly80Ala 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 half-saturating substrate concentration (S0.5) for ATP, and Thr168Pro resulted in eight- and sixfold increases in the S0.5 values for ATP and glucose, respectively. The Thr209Met and Va1226Met mutations exhibited three- and fivefold increases, respectively, in the S0.5 for ATP, whereas the Cys213Arg mutation resulted in a fivefold increase in the S0.5 for glucose. These mutations also led to a small yet significant reduction in V(maX). Of all the mutations studied, only the Cys213Arg mutation had reduced enzymatic activity and decreased thermal stability. Two mutants, Ala53Ser and Va1367Met, showed kinetic and thermal stability properties similar to those of WT. These mutants had increased sensitivities to the known negative effectors 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 kinetic alterations but also to the regulation of GK activity.