Phosphorylated thiosugars: synthesis, properties, and reactivity in enzymatic reactions.
A number of phosphorylated thiosugars have been prepared and tested as substrates for metabolic reactions. 6-Thioglucose-6-P is readily synthesized by reaction of 6-tosylglucose with trisodium thiophosphate at pH 10 in aqueous solution; the product has only sulfur between carbon and phosphorus. When ethyl glycerate is tosylated and treated similarly with thiophosphate, a 5:1 mixture of 3-thioglycerate-3-P and the 2-isomer is formed. 6-Thioglucose-6-P is converted by glycolytic enzymes to triose phosphates, 3-thioglycerol-3-P and 3-thioglycerate-3-P, and is oxidized by enzymes of the hexose monophosphate shunt to 5-thioribulose-5-P, which can be converted via phosphoribulokinase and ribulose-bis-P carboxylase into 3-P-glycerate and 3-thioglycerate-3-P. For most of the non-phosphoryl-transferring enzymes there are only moderate effects on Vmax and Km. Phosphoglucoisomerase, however, is very sensitive to the sulfur for oxygen change, with Vmax decreasing 60-fold and Km increasing 15-fold. Surprisingly, phosphoribulokinase has a V/K value for 5-thioribulose-5-P that is over 3 orders of magnitude less than for ribulose-5-P. 6-Thio-glucose-6-P was found to be a substrate for several enzymes that transfer the phosphoryl group. It is as good a substrate for alkaline phosphatase as glucose-6-P, and with phosphoglucomutase it is converted to 6-thioglucose-1-P with a rate that is 11% of the rate of reaction of glucose-1-P, with a Keq value of 45.6. The free energy of hydrolysis of the phosphorylated thiol is thus -7.2 kcal/mol at pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)
Knight
W B
WB
Institute for Enzyme Research, University of Wisconsin, Madison 53705.
Sem
D S
DS
Smith
K
K
Miziorko
H M
HM
Rendina
A R
AR
Cleland
W W
WW
D000469:Alkaline Phosphatase / Q000378:metabolism*; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism*; D006593:Hexokinase / Q000378:metabolism*; D007202:Indicators and Reagents; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010733:Phosphoglucomutase / Q000378:metabolism*; D010734:Phosphogluconate Dehydrogenase / Q000378:metabolism*; D010762:Phosphorylase a / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor); D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000138:chemical synthesis* / Q000378:metabolism; D013865:Thioglycosides / Q000138:chemical synthesis* / Q000378:metabolism
D007202:Indicators and Reagents; D013403:Sugar Phosphates; D013865:Thioglycosides; D010734:Phosphogluconate Dehydrogenase; D005954:Glucosephosphate Dehydrogenase; D010762:Phosphorylase a; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); D006593:Hexokinase; C022390:phosphoribulokinase; D000469:Alkaline Phosphatase; D010733:Phosphoglucomutase