Biochemical, physiological, and molecular characterization of sucrose synthase from Daucus carota.
Sucrose synthase (EC 2.4.1.13) from carrot (Daucus carota) is a tetramer with a molecular mass of 320 kD and subunits of 80 kD. The enzyme has a pH optimum of 7.0 (cleavage direction). Maximal activities were measured at 55 degrees C. The Km for Suc was estimated as 87 mM and for UDP as 0.39 mM. Fructose acts as a noncompetitive inhibitor with an inhibition constant of 17.2 mM. In contrast, glucose inhibits carrot sucrose synthase uncompetitively with an inhibition constant of 4.3 mM. cDNA clones encoding a single class of sucrose synthase polypeptide were isolated and sequenced. DNA gel blot analysis also indicated the occurrence of only one to two genes. The deduced amino acid sequence of the carrot enzyme is highly homologous to the sucrose synthase sequences of tomato, potato, and bean. A comparison of the cDNA-derived amino acid sequence with the SS1- and SS2-type sucrose synthase sequences of the monocot plants maize, rice, and barley showed that the carrot enzyme is neither of the SS1 nor of the SS2 type. High enzyme activity was found in roots and petioles of developing carrot plants, with maximal activity in roots at the transition of primary roots to tap roots. Enzyme activity was highly correlated with both polypeptide and transcript levels, indicating that gene expression is regulated mainly at the mRNA level in the different tissues and organs of developing carrot plants.
Sebková
V
V
Friedrich Miescher-Institut, Basel, Switzerland.
Unger
C
C
Hardegger
M
M
Sturm
A
A
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018552:Daucus carota / Q000201:enzymology* / Q000254:growth & development; D004591:Electrophoresis, Polyacrylamide Gel; D005632:Fructose / Q000494:pharmacology; D015870:Gene Expression; D015723:Gene Library; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012275:Oryza / Q000201:enzymology; D010802:Phylogeny; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D018749:RNA, Plant / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D003313:Zea mays / Q000201:enzymology
D012333:RNA, Messenger; D018749:RNA, Plant; D005632:Fructose; D005964:Glucosyltransferases; C022699:sucrose synthase