Fructokinase activity in the sugarcane culm : expression patterns and kinetic properties

Hoepfner, Suzanne Wilmien (2001-03)

Thesis (MSc)--University of Stellenbosch, 2001.

Thesis

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.

AFRIKAANSE OPSOMMING: Vyf heksokinases, twee fruktokinases en drie heksokinases, is in suikerrietstingel ge'ldentifiseer. Fruktokinase, In fruktose-spesifieke heksose fosforileringsensiem, is verder ondersoek. Twee isovorme, FRK1 en FRK2, is gevind. Die isovorme is gesuiwer tot homogeniteit en teenliggarne is teen beide vervaardig. FRK1 en FRK2 het albei In pH optimum van 8.0 en beide is homodimere van 69 kDa, bestaande uit subeenhede van 33 kDa. FRK2 is baie gevoelig vir substraatremming deur fruktose, terwyl FRK1 nie gerem word deur konsentrasies selfs so hoog as 1.0 mM fruktose nie. Suikerriet FRK2 is meer sensitief vir substraatremming as FRK2 van ander plante. Die reaksie wat deur FRK1 gekataliseer word, is ATP-spesifiek. Die FRK2-reaksie kan In verskeidenheid nukleotiedtrifosfate benut en geen substraatremming kom voor wanneer die reaksie gemeet word met UTP in plaas van ATP nie. Gebaseer op die bevindinge word "n model voorgestel waar twee nukleotiedtrifosfaatbindingsetels op die ensieme voorkom. Een van die setels is In ATP-spesifieke regulatoriese setel, terwyl die ander In katalitiese setel met bree substraatspesifisiteit is. Verder postuleer ons twee fruktose-bindingsetels. Een van die setels is katalities en die ander een is 'n allosteriese regulatoriese setel. Binding van fruktose aan die allosteriese setel kan net plaasvind as ATP gebind is in die ATP-regulatoriese setel. So 'n konformasie kan die kinetiese eienskappe van FRK2 verduidelik. Die uitdrukking van fruktokinase-proteren en die totale fruktokinase-aktiwiteit neem beide af gedurende ontwikkeling. Gevolglik is die afname in aktiwiteit die gevolg van verminderde protelenuitdrukkinq en nie inaktivering van bestaande proteten nie. Die verhouding van FRK1- tot FRK2-aktiwiteit is afhanklik van die ontwikkelingstadium van die weefsel. Dit wil voorkom of FRK1 die isovorm is wat by voorkeur in volwasse weefsel uitgedruk word. Vorige bepalings van fruktokinase-aktiwiteit in ru-ekstrakte was onakkuraat as gevolg van die uiteenlopende kinetiese eienskappe van die isovorme. Gebaseer op die bevindinge van hierdie projek stel ons In oorspronklike metode voor waarvolgens beide die aktiwiteit van elke isovorm en totale fruktokinase-aktiwiteit akkuraat bereken kan word met behulp van 'n wiskundige formule.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/52276
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