A kinetic study of sugarcane sucrose synthase.
The kinetic data on sugarcane (Saccharum spp. hybrids) sucrose synthase (SuSy, UDP-glucose: D-fructose 2-alpha-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. 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.
Institute for Plant Biotechnology, University of Stellenbosch, South Africa. wolfgang@azargen.com
Wolfgang E
WE
Schäfer
Johann M
JM
Rohwer
Frederik C
FC
Botha
D001667:Binding, Competitive; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D005632:Fructose / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D007527:Isoenzymes; D007700:Kinetics; D008954:Models, Biological; D018515:Plant Leaves / Q000201:enzymology; D031786:Saccharum / Q000201:enzymology*; D013379:Substrate Specificity; D013395:Sucrose / Q000737:chemistry / Q000378:metabolism; D014530:Uridine Diphosphate / Q000737:chemistry / Q000378:metabolism; D014532:Uridine Diphosphate Glucose / Q000737:chemistry / Q000378:metabolism
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D005632:Fructose; D013395:Sucrose; D014530:Uridine Diphosphate; D005964:Glucosyltransferases; C022699:sucrose synthase; D014532:Uridine Diphosphate Glucose