Donor substrate regulation of transketolase

Abstract

The influence of substrates on the interaction of apotransketolase with thiamin diphosphate was investigated in the presence of magnesium ions. It was shown that the donor substrates, but not the acceptor substrates, enhance the affinity of the coenzyme either to only one active center of transketolase or to both active centers, but to different degrees in each, resulting in a negative cooperativity for coenzyme binding. In the absence of donor substrate, negative cooperativity is not observed. The donor substrate did not affect the interaction of the apoenzyme with the inactive coenzyme analogue, N3¢-pyridyl-thiamin diphosphate. The influence of the donor substrate on the coenzymeapotransketolase interaction was predicted as a result of formation of the transketolase reaction intermediate 2-(a,b-dihydroxyethyl)-thiamin diphosphate, which exhibited a higher affinity to the enzyme than thiamin diphosphate 1. The enhancement of thiamin diphosphate's affinity to apotransketolase in the presence of donor substrate is probably one of the mechanisms underlying the substrate-affected transketolase regulation at low coenzyme concentrations.

Which stage of the process of apotransketolase interaction with thiamine diphosphate is affected by the regulatory activity of the donor substrate?

“…The donor substrate affects neither the stage of primary ThDP binding to apoTK, resulting in the formation of the intermediate complex TKÁÁÁThDP, nor the rate of the forward conformational transition, resulting in the formation of the catalytically active holoenzyme TK* -ThDP. The inability of the donor substrate to affect the value of k þ1 indicates that the increase in ThDP affinity for TK, observed by us previously in the presence of donor substrates (14), is due to their effect on k 71 , the rate constant of the reverse conformational transition of the catalytically active TK form (TK* -ThDP) into the inactive intermediate complex of the enzyme with ThDP (TKÁÁÁThDP) (Scheme 1). This assumption was further corroborated by the result of our experiments, in which we studied the kinetics of ThDP dissociation from the active sites of holoTK.…”

“…At the second stage (slow), associated with conformational changes of the protein molecule, the catalytically active holoenzyme is formed (TK* -ThDP in Scheme 1). It is worthwhile to note that the formation of TK* -ThDP may be followed by the change in the absorbance at the wavelength range of 300 -340 nm (6,7,14); this change characterizes the conformational changes in the active sites of TK.…”

“…Donor substrates increase the affinity of the coenzyme for TK, irrespective of whether Ca 2þ or Mg 2þ is used as a cofactor (14). Moreover, in the presence of Mg 2þ , the donor substrate induces a pronounced non-equivalence of the active sites in ThDP binding (if it has not been manifested in the absence of the substrate).…”

“…Moreover, in the presence of Mg 2þ , the donor substrate induces a pronounced non-equivalence of the active sites in ThDP binding (if it has not been manifested in the absence of the substrate). The increase in the affinity of ThDP for TK, observed in the presence of the donor substrate, is due to the formation of DHEThDP, an intermediate of the TK reaction (14), which is characterized by a higher affinity for apoTK than the coenzyme itself (15). The increase in the affinity of ThDP for apoTK in the presence of the donor substrate is a mechanism suggested to be important for the regulation of the enzyme activity by the substrate at low concentrations of the coenzyme in the cell (14).…”

“…The increase in the affinity of ThDP for TK, observed in the presence of the donor substrate, is due to the formation of DHEThDP, an intermediate of the TK reaction (14), which is characterized by a higher affinity for apoTK than the coenzyme itself (15). The increase in the affinity of ThDP for apoTK in the presence of the donor substrate is a mechanism suggested to be important for the regulation of the enzyme activity by the substrate at low concentrations of the coenzyme in the cell (14). In this work, we determined that stage within reconstitution (according to Scheme 1) where the donor substrate causes the increase in cofactor affinity.…”