Browsing by Author "Hoepfner, Suzanne Wilmien"
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- ItemFructokinase activity in the sugarcane culm : expression patterns and kinetic properties(Stellenbosch : Stellenbosch University, 2001-03) Hoepfner, Suzanne Wilmien; Botha, F. C.; Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology.ENGLISH ABSTRACT: Five hexose kinases, two fructokinases and three hexokinases, were identified in sugarcane culm. Fructokinase, a fructose specific hexose phosphorylating enzyme, was further investigated. Two isoforms, FRK1 and FRK2, were found. The isoforms were purified to homogeneity and antibodies raised against each. Both FRK1 and FRK2 have pH optima of 8.0 and both are homodimers of 69 kDa, consisting of subunits of 33 kDa. FRK2 was subject to substrate inhibition by fructose concentrations exceeding 0.1 mM while FRK1 was not inhibited by 1.0 mM fructose. Sugarcane FRK2 is more sensitive to substrate inhibition than FRK2 from other plants. The reaction catalysed by FRK1 is ATP-specific. The FRK2 reaction can utilise a variety of nucleotide triphosphates and no substrate inhibition is apparent when assayed with UTP instead of ATP. We proposed the existence of two nucleotide triphosphate binding sites on the enzymes. One of the sites is an ATP-specific regulatory site while the other is a catalytic site with wide substrate specificity. Additionally two fructose-binding sites are proposed. One is a catalytic site and the other a allosteric regulatory site. Binding of fructose to the allosteric site is only possible if ATP is present in the regulatory ATP-binding site. Such a configuration could explain the kinetic properties of FRK2. Both fructokinase protein expression and total fructokinase activity decreased during development. Consequently the decrease in activity is the result of decreased expression and not inactivation of existing protein. The ratio of FRK2 to FRK1 activity is dependent on the developmental stage of the tissue. FRK1 appears to be the isoform that is preferentially expressed in mature tissue. Previous measurements of fructokinase activity in crude extracts have been inaccurate as a result of the divergent kinetic properties of the isoforms. Based on the findings in this project a novel method is proposed whereby both the activity of each isoform and total fructokinase activity can be accurately calculated using a mathematical equation.