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.
Schäfer
Wolfgang E
WE
Institute for Plant Biotechnology, University of Stellenbosch, South Africa. wolfgang@azargen.com
Rohwer
Johann M
JM
Botha
Frederik C
FC
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