1698 DIABETES, VOL. 48, SEPTEMBER 1999 Structural Model of Human Glucokinase in Complex With Glucose and ATP Implications for the Mutants That Cause Hypo- and Hyperglycemia Bhuvaneshwari Mahalingam, Antonio Cuesta-Munoz, Elizabeth A. Davis, Franz M. Matschinsky, Robert W. Harrison, and Irene T. Weber Mutations in human glucokinase are implicated in the development of diabetes and hypoglycemia. Human glucokinase shares 54% identical amino acid residues with human brain hexokinase I. This similarity was used to model the structure of glucokinase by analogy to the crystal structure of brain hexokinase. Gluco- kinase was modeled with both its substrates, glucose and MgATP, to understand the effect of mutations. The glucose is predicted to form hydrogen bond interac- tions with the side chains of glucokinase residues Thr 168, Lys 169, Asn 204, Asp 205, Asn 231, and Glu 290, similar to those observed for brain hexokinase I. The magnesium ion is coordinated by the carboxylates of Asp 78 and Asp 205 and the -phosphate of ATP. ATP is predicted to form hydrogen bond interactions with residues Gly 81, Thr 82, Asn 83, Arg 85, Lys 169, Thr 228, Lys 296, Thr 332, and Ser 336. Mutations of residues close to the predicted ATP binding site produced dra- matic changes in the K m for ATP, the catalytic rate, and a loss of cooperativity, which confirmed our model. Mutations of residues in the glucose binding site dra- matically reduced the catalytic activity, as did a muta- tion that was predicted to disrupt an -helix. Other mutations located far from the active site gave smaller changes in kinetic parameters. In the absence of a crys- tal structure for glucokinase, our models help ratio- nalize the potential effects of mutations in diabetes and hypoglycemia, and the models may also facilitate the discovery of pharmacological glucokinase activa- tors and inhibitors. Diabetes 48:1698–1705, 1999 H uman hexokinase IV, which is usually called glucokinase (ATP:D-hexose 6-phosphotrans- ferase, EC 2.7.1.1), is a 50-kD enzyme that cat- alyzes the ATP-dependent phosphorylation of glucose to glucose-6-phosphate as the first step, and the first rate-limiting step, in the glycolytic pathway. The family of related enzymes, glucokinase and hexokinases I, II, and III, mediate glucose phosphorylation in mammalian tissues. Glucokinase is characterized by a high S 0.5 for glucose of 5–8 mmol/l compared with K m values of 20–130 μmol/l for human hexokinases I–III and by a lack of product inhibition by glucose-6-phosphate compared with the hexokinases. At physiological levels, glucokinase is virtually inactive with man- nose and fructose in contrast to hexokinases I–III, hence the common usage of “glucokinase” rather than hexokinase IV. Glucokinase has an important role as glucose sensor and metabolic signal generator in pancreatic -cells and hepato- cytes (1–3). A small change in the activity or glucose affinity of glucokinase due to mutation will displace the threshold for insulin secretion in response to glucose from its normal phys- iological setting of 5 mmol/l. Mutations in the glucokinase gene can lead to development of an autosomal dominant form of type 2 diabetes (4–6). Nearly 100 of these mutations have been identified. Gluco- kinase mutations associated with diabetes show a variety of kinetic defects, including reduced catalytic activity, increased K m for glucose (7,8), and/or increased K m for ATP (9). Other mutants show reduced thermal stability (9). Recently, a glucokinase mutation was identified in a family with hyper- insulinemia; the autosomally transmitted V455M mutant shows a decreased S 0.5 for glucose (10,11). Previously, the structure of human glucokinase with glu- cose was modeled by analogy to the crystal structure of yeast hexokinase (12–14). Yeast hexokinase is a paradigm for catalysis by induced fit: a large conformational change occurs on binding of glucose, as illustrated by two different crystal structures (15). Hexokinase folds into two domains, with the active site lying in a cleft between the two domains. The B isozyme of hexokinase was crystallized with the inhibitor, o-toluoylglucosamine (OTG), and the structure is in an “open” conformation in which the two domains are further apart (16). Yeast hexokinase A was crystallized with glucose From the Department of Microbiology and Immunology (B.M., R.W.H., I.T.W.), Thomas Jefferson University; and the Department of Biochemistry and Biophysics and the Diabetes Research Center (A.C.-M., E.A.D., F.M.M.), University of Pennsylvania, Philadelphia, Pennsylvania. Address correspondence and reprint requests to Irene T. Weber, Depart- ment of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, 233 South 10th St., Philadelphia, PA 19107. E-mail: weber@asterix.jci.tju.edu. Received for publication 23 February 1999 and accepted in revised form 12 May 1999. R.W.H. and I.T.W. jointly hold shares in Merck Corporation and Glaxo- Welcome. GST, glutathione S-transferase; rms, root mean square.