Isozymes of plant hexokinase: occurrence, properties and functions.

Hexokinase (HK) occurs in all phyla, as an enzyme of the glycolytic pathway. Its importance in plant metabolism has emerged with compelling evidence that its preferential substrate, glucose, is both a nutrient and a signal molecule that controls development and expression of different classes of genes. A variety of plant tissues and organs have been shown to express multiple HK isoforms with different kinetic properties and subcellular localizations. Although plant HK is known to fulfill a catalytic function and act as a glucose sensor, the physiological relevance of plural isoforms and their contribution to either function are still poorly understood. We review here the current knowledge and hypotheses on the physiological roles of plant HK isoforms that have been identified and characterized. Recent findings provide hints on how the expression patterns, biochemical properties and subcellular localizations of HK isoforms may relate to their modes of action. Special attention is devoted to kinetic, mutant and transgenic data on HKs from Arabidopsis thaliana and the Solanaceae potato, tobacco, and tomato, as well as HK gene expression data from Arabidopsis public DNA microarray resources. Similarities and differences to known properties of animal and yeast HKs are also discussed as they may help to gain further insight into the functional adaptations of plant HKs.

The futile cycling of hexose phosphates could account for the fact that hexokinase exerts a high control on glucose phosphorylation but not on glycolytic rate in transgenic potato (Solanum tuberosum) roots.

The metabolism of potato (Solanum tuberosum) roots constitutively over- and underexpressing hexokinase (HK, EC 2.7.1.1) was examined. An 11-fold variation in HK activity resulted in altered root growth, with antisense roots growing better than sense roots. Quantification of sugars, organic acids and amino acids in transgenic roots demonstrated that the manipulation of HK activity had very little effect on the intracellular pools of these metabolites. However, adenylate and free Pi levels were negatively affected by an increase in HK activity. The flux control coefficient of HK over the phosphorylation of glucose was measured for the first time in plants. Its value varied with HK level. It reached 1.71 at or below normal HK activity value and was much lower (0.32) at very high HK levels. Measurements of glycolytic flux and O(2) uptake rates demonstrated that the differences in glucose phosphorylation did not affect significantly glycolytic and respiratory metabolism. We hypothesized that these results could be explained by the existence of a futile cycle between the pools of hexose-Ps and carbohydrates. This view is supported by several lines of evidence. Firstly, activities of enzymes capable of catalyzing these reactions were detected in roots, including a hexose-P phosphatase. Secondly, metabolic tracer experiments using (14)C-glucose as precursor showed the formation of (14)C-fructose and (14)C-sucrose. We conclude that futile cycling of hexose-P could be partially responsible for the differences in energetic status in roots with high and low HK activity and possibly cause the observed alterations in growth in transgenic roots. The involvement of HK and futile cycles in the control of glucose-6P metabolism is discussed.

Cloning, expression, purification, and properties of a putative plasma membrane hexokinase from Solanum chacoense.

A full-length hexokinase cDNA was cloned from Solanum chacoense, a wild relative of the cultivated potato. Analysis of the predicted primary sequence suggested that the protein product, ScHK2, may be targeted to the secretory pathway and inserted in the plant plasma membrane, facing the cytosol. ScHK2 was expressed as a hexahistidine-tagged protein in Escherichia coli. Expression conditions for this construct were optimized using a specific anti-hexokinase polyclonal anti-serum raised against a truncated version of ScHK2. The full-length recombinant protein was purified to electrophoretic homogeneity using immobilized metal ion affinity chromatography followed by anion exchange chromatography on Fractogel EMD DEAE-650 (S). The purified enzyme had a specific activity of 5.3 micromol/min/mg protein. Its apparent Kms for glucose (23 microM), mannose (30 microM), fructose (5.2 mM), and ATP (61 microM) were in good agreement with values found in the literature for other plant hexokinases. Hexahistidine-tagged ScHK2 was highly sensitive to pH variations between 7.7 and 8.7. It was inhibited by ADP and insensitive to glucose-6-phosphate. These findings constitute the first kinetic characterization of a homogeneous plant hexokinase preparation. The relevance of ScHK2 kinetic properties is discussed in relation to the regulation of hexose metabolism in plants.