A kinetic study of sugarcane sucrose synthase

Authors


  • Enzyme: sucrose synthase (EC 2.4.1.13).

W. E. Schäfer, Institute for Plant Biotechnology, University of Stellenbosch, Private Bag X1, 7602 Matieland, South Africa. Fax: +27 21 8083835, Tel.: +27 21 8083834,
E-mail: wolfgang@azargen.com

Abstract

The kinetic data on sugarcane (Saccharum spp. hybrids) sucrose synthase (SuSy, UDP-glucose: d-fructose 2-α-d-glucosyltransferase, EC 2.4.1.13) are limited. We characterized kinetically a SuSy activity partially purified from sugarcane variety N19 leaf roll tissue. Primary plot analysis and product inhibition studies showed that a compulsory order ternary complex mechanism is followed, with UDP binding first and UDP-glucose dissociating last from the enzyme. Product inhibition studies showed that UDP-glucose is a competitive inhibitor with respect to UDP and a mixed inhibitor with respect to sucrose. Fructose is a mixed inhibitor with regard to both sucrose and UDP. Kinetic constants are as follows: Km values (mm, ± SE) were, for sucrose, 35.9 ± 2.3; for UDP, 0.00191 ± 0.00019; for UDP-glucose, 0.234 ± 0.025 and for fructose, 6.49 ± 0.61. inline image values were, for sucrose, 227 mm; for UDP, 0.086 mm; for UDP-glucose, 0.104; and for fructose, 2.23 mm. Replacing estimated kinetic parameters of SuSy in a kinetic model of sucrose accumulation with experimentally determined parameters of the partially purified isoform had significant effects on model outputs, with a 41% increase in sucrose concentration and 7.5-fold reduction in fructose the most notable. Of the metabolites included in the model, fructose concentration was most affected by changes in SuSy activity: doubling and halving of SuSy activity reduced and increased the steady-state fructose concentration by about 42 and 140%, respectively. It is concluded that different isoforms of SuSy could have significant differential effects on metabolite concentrations in vivo, therefore impacting on metabolic regulation.

Ancillary