Abstract
Sugar kinases are enzymes known to catalyze the phosphorylation of different sugar substrates. The sugar kinases encompassed by the ROK (repressor, open reading frame, kinase) superfamily offer a unique opportunity to explore the evolutionary origins of enzyme specificity. We probed evolutionary events that occur during the optimization of glucokinase activity in two members of the ROK superfamily, Alsk and NanK, which share only 21% sequence identity with one another. Following random mutagenesis and in vivo functional selection, we identified two structurally overlapping mutational “hot spots” in the sugar kinase scaffold. Steady state kinetic analyses of the selected variants demonstrate that the native activities of AlsK and NanK are largely unaffected by the glucokinase-enhancing substitutions. Furthermore, the variants have acquired an increased ability to phosphorylate a variety of nonnatural carbohydrate substrates as a result of the evolutionary process. This finding is consistent with an evolutionary process that includes the formation of intermediates possessing relaxed substrate specificities during the initial steps of enzyme functional divergence. Highly evolved enzymes display low Km values for their substrates as a result of their functional refinement toward increased specificity. An anomalous behavior of higher order enzymes is presented by human glucokinase, a monomeric enzyme possessing kinetic cooperativity and a high glucose Km value. The sigmoidal response of the turnover rate as a function of glucose concentration allows human pancreatic glucokinase to regulate glucose concentration in the blood stream. The importance of glucokinase proper function is reflected in the different diseases associated with genetic mutations in glk. Maturity onset diabetes of young (MODY) is associated with genetic lesions in glk that reduce catalytic activity, while persistent hyperinsulinemia of infancy (PHHI) is found in hyperactive glucokinase variants. Different mechanisms have been postulated to explain glucokinase kinetic cooperativity, several of which involve the existence of multiple enzyme species interconverting with a rate slower than catalysis. Our kinetic studies of glucose binding, obtained via stopped-flow, suggest the existence of at least two species in equilibrium in the absence of glucose, which are able to bind glucose and subsequently isomerize. This postulated mechanism was further tested experimentally by analytical ultracentrifugation (AUC) and nuclear magnetic resonance (NMR). Analytical ultracentrifugation results show that human pancreatic glucokinase is a monomer in solution with a sedimentation coefficient of 3.5 S. Unfortunately, AUC does not provide the resolution needed to separate the different conformations of human glucokinase. Preliminary xii xiii HSQC-NMR spectra are characteristic of either (a) a partially unfolded protein or (b) multiple species slowly interconverting. Additional investigations conducted on AUC eliminated the possibility of aggregates as an explanation for the obsvered NMR spectra. Future experiments to test the existence and rates of GK conformers in solution are discussed. Structurally, glucose binding to human pancreatic glucokinase involves large conformational changes of the small domain, towards formation of a more compact structure. Of particular interest was the secondary structural element located at the C-terminus (helix α13), which, upon ligand binding, moves from a solvent exposed position to a hydrophobic pocket. Deletion of helix α13 abolishes cooperativity and restores Michaelis-Menten kinetics. Addition of a thirteen amino acid synthetic peptide in trans does not restore the kcat value of the truncated variant. Elongation of α13 via the addition of a C-terminal polyalanine tail does not affect the glucokinase steady-state kinetics. Randomization studies of residues 450-456 of helix α13, followed by selections in a glucokinase-deficient bacterium BM5340(DE3), identified variants that possessed lower K0.5 glucose values, Hill coefficient near unity and enhanced equilibrium binding affinity for glucose. Our studies on helix α13 demonstrate the essential role played by this structural element in governing cooperativity and establish a link between its primary amino acid sequence and the functional dynamics of the glucokinase scaffold that are required for allostery.
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