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Conversion of Glucokinase to an Enzyme with High Affinity for Glucose Moulay A

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Conversion of Glucokinase to an Enzyme with High Affinity for Glucose Moulay A Study of the Regulatory Properties of Glucokinase by Site-Directed Mutagenesis Conversion of Glucokinase to an Enzyme With High Affinity for Glucose Moulay A. Moukil, Maria Veiga-da-Cunha, and Emile Van Schaftingen To identify the amino acids involved in the specific reg- ulatory properties of glucokinase, and particularly its low affinity for glucose, mutants of the human islet here is now overwhelming evidence that gluco- enzyme have been prepared, in which glucokinase-spe- kinase, the enzyme that phosphorylates glucose cific residues have been replaced. Two mutations in the liver and in pancreatic islets, plays a critical increased the affinity for glucose by twofold (K296M) role as a glucose-sensing device (1). This evidence, and sixfold (Y214A), the latter also decreasing the Hill T originally derived from studies on the control of glucose c o e fficient from 1.75 to 1.2 with minimal change in the a ffinity for AT P. Combining these two mutations with metabolism in the liver and in islets (2), has now received N166R resulted in a 50-fold decrease in the half-satu- strong support with the discovery that a loss of glucokinase ac t i v i t y , due to either mutations in the glucokinase gene in rating substrate concentration (S0 . 5) value, which became then comparable to the Km of hexokinase II. The humans (3–5) or gene knock-out in animal models (6,7), location of N166, Y214, and K296 in the three-dimen- results in diabetes. Another strong piece of evidence in favor sional structure of glucokinase suggests that these of glucokinase being the glucose sensor in -cells is the find - mutations act by favoring closure of the catalytic cleft. ing that an autosomal-dominant form of hyperinsulinemia is As a rule, mutations changed the affinity for glucose and linked to a mutation of glucokinase that increases the affinity for the competitive inhibitor mannoheptulose (MH) in of this enzyme for glucose by about threefold (8). parallel, whereas they barely affected the affinity for The role played by glucokinase as a glucose sensor is due N-acetylglucosamine (NAG). These and other results suggest that NAG and MH bind to the same site but to to its specific regulatory properties, mainly a low affinity for d i fferent conformations of glucokinase. A small reduc- glucose, with a sigmoidal saturation curve for this substrate, tion in the affinity for the regulatory protein was and a lack of inhibition by physiological concentrations of glu- observed with mutations of residues on the smaller cose-6-phosphate (G-6-P) (9–11). These properties are in domain and in the hinge region, confirming the bipartite sharp contrast with those of homologous enzymes such as the nature of the binding site for the regulatory protein. mammalian low-Km hexokinases. In addition, glucokinase The K296M mutant was found to have a threefold has, compared with other hexokinases, the unique property decreased affinity for palmitoyl CoA; this effect was of being inhibited by a regulatory protein and by long-chain additive to that previously observed for the E279Q acyl CoAs. Kinetic evidence indicates that these two types of mutant, indicating that the binding site for long-chain inhibitors bind to a site distinct from the catalytic site despite acyl CoAs is located on the upper face of the larger domain. D i a b e t e s 4 9 :1 9 5–201, 2000 the fact that their action is competitive with glucose (12). The properties mentioned above appear to be shared by all ani- mal glucokinases (13,14), suggesting that the amino acid residues participating in these regulatory functions have been conserved. The cDNA encoding Xe n o p u s liver glucokinase has been cloned and shown to encode a protein with 78% amino acid identity with mammalian glucokinases (15). Glucokinase- sp e c i fi c residues have then been mutated in the Xe n o p u s From the Laboratoire de Chimie Physiologique, Christian de Duve Institute of enzyme and replaced by their counterparts in the C-half of rat Cellular Pathology and Université Catholique de Louvain, Brussels, Belgium. hexokinase I, an enzyme that does not share the regulatory Address correspondence and reprint requests to Emile Van Schaftingen, Avenue Hippocrate 75, B-1200 Brussels, Belgium. E-mail: vanschaftingen@ properties of glucokinase. This approach led to the identifi- b c h m . u c l . a c . b e . cation of two sets of residues involved in the binding of the Received for publication 8 July 1999 and accepted in revised form regulatory protein, one at the tip of the smaller domain and 1 8 October 1999. another close to the hinge region (15) according to the gluco- G - 6 - P, glucose-6-phosphate; I , 50% inhibition; MH, mannoheptulose; 5 0 kinase model developed by St. Charles et al. (16). Confirm a - N A G , N-acetylglucosamine; PCR, polymerase chain reaction, S0 . 5, half-sat- urating substrate concentration. tion of this localization came from other experiments per- DIABETES, VOL. 49, FEBRUARY 2000 195 GLUCOKINASE REGULATORYPROPERTIES TA B L E 1 Primers used for preparation of the mutants M u t a n t P r i m e r s Diagnostic restriction site K 1 6 1 Q 5 -ca g g g c a t c c t t c t c a a c t g g a c c a a g g c c - 3 + S a u3 A I 5 - a t c g a t g t c t t c g t g c c t c a c a g g a a a g g a - 3 N180D, G183S (G) 5 - g t gt cg c t t c t g c g a g a c g c t a t c a a a c g g - 3 5 - g a c a tcg t t c c c t t c t g c t c c t g a g g c c t t - 3 A 2 0 8 G 5 - a c g a t g a t c t c c t g c t a c t a c g a a g a c c a t - 3 – B a lI 5 - gcc c a c c g t g t c a t t c a c c a t t g c c a c c a c - 3 Y 2 1 4 A 5 -g c gt a c g a a g a c c a t c a g t g c g a g g - 3 + R s aI 5 - g c a g g a g a t c a t c g t g g c c a c c g - 3 C 2 3 0 S 5 - tc gaatgcctgctacatggaggagatgc- 3 + Ta qI 5 - g c c c g t g c c c a c g a t c a t g c c g a c - 3 L 2 7 1 T, E272D, R275K (H) 5 - g a ca a gc t a g t g gac g a g a g c t c t g c a a a c - 3 + M a eI 5 - a t agt c cg tc a g g a a c t c g t c c a g c t c g c c - 3 Y 2 8 9 F, G294S (I) 5 - c t c a t aag t g g c a a g t a c a t g g g c g a g c t g - 3 + Ta qI 5 - c t t c t cg aa c a g c t g c t g a c c g g g g t t t g c - 3 K 2 9 6 M 5 - atg t a c a t g g g c g a g c t g g t g c g g - 3 + N s p HI 5 - g c c a c c t a t g a g c t t c t c a t a c a g - 3 L304N, R308D, V310T, E312K, N313G (J) 5 -a cg g a caa ag gc c t g c t c t t c c a c g g g - 3 + Ta qI 5 - g a gg t cg a g c a g c a c at tc c g c a c c a g c t c - 3 Mutated nucleotides are underlined. The diagnostic restriction sites are also shown. formed on human islet glucokinase where it was found that cated, 25 mmol/l HEPES, pH 7.1, 25 mmol/l KCl, 2.5 mmol/l MgCl2, 0.6 mm o l / l all mutations that decreased the affinity of glucokinase for its NA D +, 1 mmol/l ATP-Mg, the indicated glucose concentrations, and 10 µg/ml regulatory protein clustered in the hinge region and nearby Leuconostoc mesenteroides G-6-P dehydrogenase. The inhibition by palmitoyl CoA was tested with no excess of Mg2+ over ATP , to avoid partial inhibition (20) pos- in the larger and the smaller domains (17). sibly due to the lowering of the critical micelle concentration. The effects of the The goal of the present work was to extend these muta- regulatory protein and the phosphorylation of substrates other than glucose genesis studies, using the same rationale as described above were tested with the pyruvate kinase/lactate dehydrogenase–coupled assay pre- (15), to identify additional residues involved in the regulatory viously described (18).
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