Messung der T-Fixierung bei einigen Teilschritten der Glykolyse

The pyruvate kinase reaction occurs in separate phosphate- and proton-transfer stages: (formula; see text) K+, Mg2+, and Mg.ADP are known to be required for the phosphoryl transfer step, and K+ and Mg2+ with allosteric stimulation by MgATP are important for proton transfer. This paper uses the isotope trapping method with 3H-labeled water to identify the proton donor and determine when in the sequence of the catalytic cycle it is generated. When the enzyme was allowed to exchange briefly with 3H2O (pulse phase) and then diluted into a mixture containing PEP, ADP, and the cofactor K+, Mg2+, or Co2+ in D2O (chase phase), an amount of [3H]pyruvate was formed in great excess of the amount expected from steady-state catalysis in the diluted 3H-labeled water. With K+, Mg2+, and ADP at pH 6-9.5 in the pulse phase, a limit of 1.25 enzyme equiv of 3H were trapped. The concentration of PEP required for half-maximum trapping was 14-fold greater than its steady-state Km. Therefore, the rate constant for dissociation of the donor proton is estimated to be 14 times the steady-state rate of [3H]pyruvate formation, approximately 109 s-1, or 1500 s-1. At pD 6.4, Mg2+ and ADP were required in the chase, indicating that the ADP in the pulse was not bound tightly enough to be used in the chase. At pD 9.4, ADP was not required in the chase, only Mg2+ or Co2+, making it possible to limit the chase to one turnover from hybrid labeled complexes such as E.K.Mg.CoADP or E.K.Co.MgADP and PEP.(ABSTRACT TRUNCATED AT 250 WORDS)

Section: Resultssupporting

confidence: 84%

Substrate proton of the pyruvate kinase reaction

Rose¹,

Kuo²

1989

“…It is doubtful if less than 10% of transfer would have been detected. Simon and his collaborators (Simon et al, 1968), using o-[3-3H]glucose, hexokinase, phosphoglucose isomerase, phosphofructokinase, and aldolase to prepare specifically labeled dihydroxyacetone phosphate in situ, reported that some 10% of the 3H in dihydroxyacetone phosphate was transferred intramolecularly to glyceraldehyde phosphate in the isomerase-catalyzed reaction. The validity of this experiment depends, inter alia, on the isotopic purity of the tritiated glucose, and Dr. Simon has recently informed us that because some 5% of the 3H label on the starting glucose was in the 6 position, the revised estimate of the extent of tritium transfer (from dihydroxyacetone phosphate to glyceraldehyde phosphate) in the yeast isomerase-catalyzed reaction is about 5% at pH 8.7.…”

Section: Discussionmentioning

confidence: 99%

Energetics of triosephosphate isomerase: the fate of the 1(R)-³H label of tritiated dihydroxyacetone phosphate in the isomerase reaction

Herlihy¹,

Maister²,

Albery³

et al. 1976

The isomerization of specifically tritium-labeled [ 1 (R)-3H]dihydroxyacetone phosphate to D-glyceraldehyde 3-phosphate, catalyzed by the enzyme triosephosphate isomerase, has been studied. The distribution of the 3H label amongst the three possible sites (in [ 1 (R)-3Hldihydroxyace-tone phosphate, in ~-[2-~H]glyceraldehyde 3-phosphate, and in the solvent) has been followed as a function of the extent of the reaction. It is shown that the extent of transfer of the 3H label from the substrate dihydroxyacetone phosphate to Dglyceraldehyde 3-phosphate is between 3 and 6% (depending upon the extent of the reaction). The enzymic base responsible Tiosephosphate isomerase catalyzes the interconversion of the two triose phosphates, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The crystalline enzyme from chicken muscle is a dimer of identical subunits each of molecular weight about 26 000 (Putman et a]., 1972). It has no cofactor or specific metal ion requirements. As an isomerase, with a single substrate and a single product, it is particularly amenable to mechanistic study, and the equilibrium constant for the reaction is close enough to unity to allow the reaction to be run in either direction, utilizing the appropriate dehydrogenases as coupling enzymes (a-glycerophosphate dehydrogenase to remove dihydroxyacetone phosphate, or glyceraldehyde-phosphate dehydrogenase to remove glyceraldehyde phosphate). In the presence of excess of the appropriate dehydrogenase and cofactor, the reaction can be studied under conditions such that the isomerase step is rate limiting (Plaut and Knowles, 1972).Aside from these attractive features, this system presents a rare opportunity for crystallographic and spectroscopic study. With most enzymes that use two or more substrates in either direction, even if one of these is water, it is currently impossible to study productive enzyme-substrate complexes directly by crystallographic methods. approaches to the problem of the structure of enzyme-substrate complexes have had to rely on the structures of enzyme-inhibitor complexes, or "dead-end" binary complexes or abortive ternary complexes \lor two substrate systems). From such data, one has to try to deduce the structure of enzyme with real substrate bound (see, e.g., Blake et al., 1967; Steitz et al., 1969). For an isomerase, however, it is possible to find the structure both of native enzyme and of an enzyme-substrate complex. Progress with each for proton abstraction from substrate is, therefore, in almost complete isotopic equilibrium with the solvent. The remaining substrate after partial reaction increases in specific radioactivity as the reaction proceeds, showing that the preferential reaction of ' H substrate is more important than the washing out of 3H label at the stage of the exchanging intermediate. Quantitatively, these results provide the data for the first steps in the analysis described in the previous paper (Albery, W. J., and Knowles, J. R. (1976), Biochemistry, preceding paper in this issue). of these probl...

“…Xhe triose-P isomerase catalyzed reaction is generally believed to proceed through a ris-enediol intermediate as shown in Scheme I,1 i.e., a simple proton abstraction and transfer by a single base believed to be a glutamate. Proton, rather than hydride, transfer is strongly inferred in the TIM reaction from the observation that the substrate-derived tritium is largely exchanged for a medium proton (Reider & Rose, 1959; Simon et al, 1968;Maister et al, 1976) and by the slow formation of Pj and methylglyoxal, indicative of an enediol-P on the enzyme (Browne et al, 1976;Richard, 1984).…”

mentioning

confidence: 99%

Proton diffusion in the active site of triosephosphate isomerase

Rose¹,

Fung²,

Warms³

1990

The current model for hydrogen flow in the aldose-ketose isomerases is probably incorrect. Enzymes of this class are characterized by both hydrogen transfer and proton exchange in the interconversion of substrate and product. The transfer is believed to be due to the action of a unique basic residue in the active site. Exchange is presumed to occur by dissociation of the abstracted proton and reassociation from the medium prior to its transfer to the intermediate enediol on the way to product. Dissociation of a necessary proton from the intermediate state imposes limits on the overall catalytic rate depending on the pKa of the protonated base and the pH of the medium. A case in point is triose-P isomerase (TIM), where kcat is approximately 10(4) s-1. T-Labeled substrate is found to lose approximately 95% of its T to the medium when totally converted to product. Although the active site base is believed to be a glutamate of pKa = 3.9, the pH dependence of maximum velocity is known to be flat up to pH 10. The loss of hydrogen required to form product as indicated by isotope exchange must be restored completely at this high pH, requiring a base of very high pKa, or there must be some other explanation for the loss of isotope. The present study demonstrates the existence of a single proton on human and rabbit TIM and three protons on yeast TIM that rapidly exchange with the abstracted proton at the E.enediol state internal exchange. Exchange with the medium external exchange occurs from the enzyme after substrate or product has dissociated.(ABSTRACT TRUNCATED AT 250 WORDS)