It was recently described that the α5 and the α13 helices of human pancreatic glucokinase play a major role in the allosteric regulation of the enzyme. In order to understand the structural importance of these helices, we have performed site-directed mutagenesis to generate glucokinase derivatives with altered residues. We have analyzed the kinetic parameters of these mutated forms and compared them with wild-type and previously defined activating mutations in these helices (A456V and Y214C). We found two new activating mutations, A460R and Y215A, which increase the affinity of the enzyme for glucose. Our results suggest that substitutions in the α5 or the α13 helices that favor the closed, active conformation of the enzyme, either by improving the interaction with surrounding residues or by improving the flexibility of the region defined by these two helices, enhance the affinity of the enzyme for glucose, and therefore its performance as a glucose phosphorylating enzyme.This work has been financed by the Research and Technological Development Project (QLG1-CT-2001-01488) funded by the European Commission and the Spanish Ministry of Education and Science grant BMC2002-00208 to P.S. This work has also been supported by the Instituto de Salud Carlos III Network grants RCMN (C03/08) to P.S., A.L.C.M., and F.C.S., and RGDM (G03/212) to P.S. L.P. is supported by a predoctoral I3P fellowship from CSIC. A.L.C.M. was supported by Spanish Ministry of Science and Technology grant no. CYT-836, by a Novo-Nordisk Pharma grant, by SAF2001/3623, and by the FIS of the Instituto de Salud Carlos III, Madrid, Spain, grant no. 02/PI021473. F.C.S. was also supported by FIS 04/883.Peer reviewed