scholarly journals Specificity and kinetics of triose phosphate isomerase from chicken muscle

1972 ◽  
Vol 129 (2) ◽  
pp. 301-310 ◽  
Author(s):  
Sylvia J. Putman ◽  
A. F. W. Coulson ◽  
I. R. T. Farley ◽  
B. Riddleston ◽  
J. R. Knowles
Keyword(s):  
Triose Phosphate Isomerase ◽  
Proton Exchange ◽  
The Other ◽  
Chicken Breast ◽  
Dihydroxyacetone Phosphate ◽  
Exchange Reactions ◽  
Hydrogen Atoms ◽  
Triose Phosphate ◽  
Chicken Muscle ◽  
Kinetics Of

The isolation of crystalline triose phosphate isomerase from chicken breast muscle is described. The values of kcat. and Km for the reaction in each direction were determined from experiments over wide substrate-concentration ranges, and the reactions were shown to obey simple Michaelis–Menten kinetics. With d-glyceraldehyde 3-phosphate as substrate, kcat. is 2.56×105min-1and Km is 0.47mm; with dihydroxyacetone phosphate as substrate, kcat. is 2.59×104min-1and Km is 0.97mm. The enzyme-catalysed exchange of the methyl hydrogen atoms of the ‘virtual substrate’ monohydroxyacetone phosphate with solvent2H2O or3H2O was shown. This exchange is about 104-fold slower than the corresponding exchange of the C-3 hydrogen of dihydroxyacetone phosphate. The other deoxy substrate, 3-hydroxypropionaldehyde phosphate, was synthesized, but is too unstable in aqueous solution for analogous proton-exchange reactions to be studied.

On the three-dimensional structure and catalytic mechanism of triose phosphate isomerase

1981 ◽  
Vol 293 (1063) ◽  
pp. 159-171 ◽  
Keyword(s):  
Catalytic Mechanism ◽  
Polypeptide Chain ◽  
Three Dimensional ◽  
Triose Phosphate Isomerase ◽  
Dimensional Structure ◽  
Dihydroxyacetone Phosphate ◽  
Triose Phosphate ◽  
Independent Determination ◽  
Chicken Muscle

Triose phosphate isomerase is a dimeric enzyme of molecular mass 56000 which catalyses the interconversion of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate. The crystal structure of the enzyme from chicken muscle has been determined at a resolution of 2.5 A, and an independent determination of the structure of the yeast enzyme has just been completed at 3 A resolution. The conformation of the polypeptide chain is essentially identical in the two structures, and consists of an inner cylinder of eight strands of parallel |3-pleated sheet, with mostly helical segments connecting each strand. The active site is a pocket containing glutamic acid 165, which is believed to act as a base in the reaction. Crystallographic studies of the binding of DHAP to both the chicken and the yeast enzymes reveal a common mode of binding and suggest a mechanism for catalysis involving polarization of the substrate carbonyl group.

scholarly journals Enzyme-substrate and enzyme-inhibitor complexes of triose phosphate isomerase studied by 31P nuclear magnetic resonance

1979 ◽  
Vol 179 (3) ◽  
pp. 607-621 ◽  
Author(s):  
I D Campbell ◽  
R B Jones ◽  
P A Kiener ◽  
S G Waley
Keyword(s):  
Enzyme Inhibitor ◽  
Ph Dependence ◽  
Triose Phosphate Isomerase ◽  
Dihydroxyacetone Phosphate ◽  
Ligand Complex ◽  
Triose Phosphate ◽  
Kinetic Information ◽  
Transition State Analogue ◽  
Chicken Muscle ◽  
Proton Uptake

The complex formed between the enzyme triose phosphate isomerase (EC 5.3.1.1.), from rabbit and chicken muscle, and its substrate dihydroxyacetone phosphate was studied by 31P n.m.r. Two other enzyme-ligant complexes examined were those formed by glycerol 3-phosphate (a substrate analogue) and by 2-phosphoglycollate (potential transition-state analogue). Separate resonances were observed in the 31P n.m.r. spectrum for free and bound 2-phosphoglycollate, and this sets an upper limit to the rate constant for dissociation of the enzyme-inhibitor complex; the linewidth of the resonance assigned to the bound inhibitor provided further kinetic information. The position of this resonance did not vary with pH but remained close to that of the fully ionized form of the free 2-phosphoglycollate. It is the fully ionized form of this ligand that binds to the enzyme. The proton uptake that accompanies binding shows protonation of a group on the enzyme. On the basis of chemical and crystallographic information [Hartman (1971) Biochemistry 10, 146–154; Miller & Waley (1971) Biochem. J. 123, 163–170; De la Mare, Coulson, Knowles, Priddle & Offord 1972) Biochem. J. 129, 321–331; Phillips, Rivers, Sternberg, Thornton & Wilson (1977) Biochem. Soc. Trans. 5, 642–647] this group is believed to be glutamate-165. On the other hand, the position of the resonance of D-glycerol 3 phosphate (sn-glycerol 1-phosphate) in the enzyme-ligand complex changes with pH, and both monoanion and dianon of the ligand bind, although dianion binds better. The substrate, dihydroxyacetone phosphate, behaves essentially like glycerol 3-phosphate. The experiments with dihydroxy-acetone phosphate and triose phosphate isomerase have to be carried out at 1 degree C because at 37 degrees C there is conversion into methyl glyoxal and orthophosphate. The mechanismof the enzymic reaction and the reasons for rate-enhancement are considered, and aspects of the pH-dependence are discussed in an Appendix.

scholarly journals Spectrophotometric studies on the interaction between triose phosphate isomerase and inhibitors

1979 ◽  
Vol 179 (3) ◽  
pp. 623-630 ◽  
Author(s):  
R B Jones ◽  
S G Waley
Keyword(s):  
Linear Regression ◽  
Glutamic Acid ◽  
Active Sites ◽  
Crystallographic Analysis ◽  
Triose Phosphate Isomerase ◽  
The Other ◽  
Histidine Residue ◽  
Triose Phosphate ◽  
Ph Values ◽  
Chicken Muscle

The binding of ligands to chicken muscle triose phosphate isomerase was studied. Changes in u.v. absorbance of the enzyme were used to measure binding, and the dissociation constant was determined over a range of pH values. The ligands were 2-phosphoglycollate and rac-glycerol 3-phosphate (only the D-isomer, sn-glycerol 1-phosphate, binds appreciably). Non-linear regression was used to fit calculated curves to the experimental points and hence to compare different models. Both active sites in the dimeric enzyme probably bound 2-phosphoglycollate, without any interaction between the sites. The results of crystallographic analysis [phillips, Rivers, Sternberg, Thornton & Wilson (1977) Biochem. Soc Trans. 5, 642–647], and experiments on the 1H, 13C and 31P n.m.r. of enzyme or 2-phosphoglycollate were combined with the present results to provide the basis for a model in which binding depends on glutamic acid-165 being protonated and on the ligant being fully ionized; additionally, binding affects the ionization of one histidine residue (probably histidine-100). The binding of the glycerol 3-phosphate, on the other hand, was independent of pH over the range pH 6.5–8.5 but decreased at lower pH values. This is explained on a model in which the binding of the monoanion of the ligand is markedly affected by the protonation of a residue in the enzyme, but the binding of the dianion is only slightly affected by this ionization.

Reactions of hydrogen atoms with hexafluoroacetone

1978 ◽  
Vol 56 (20) ◽  
pp. 2638-2645 ◽  
Author(s):  
D. W. Grattan ◽  
K. O. Kutschke
Keyword(s):  
Rate Constant ◽  
Cross Sections ◽  
The Other ◽  
Hydrogen Atoms ◽  
Kinetics Of

Attempts were made to study the kinetics of the reaction of atomic H with (CF3)2CO vapour (HFA). Atomic H was generated from H2 by mercury photosensitization in the presence of C2H4 and HFA but the system was complicated by the loss of C2H5 radicals by addition to HFA and the kinetic results were intractable. When atomic H was generated from C3H8, the kinetics again were obscured by some unidentified reaction(s) which became more important at higher [HFA]/[C3H8]. An estimate of the rate constant for the addition of H to HFA obtained at low [HFA]/[C3H8] yielded k9 = 8.5 × 105 l mol−1 s−1. Trifluoroacetaldehyde was identified with some reliability but many of the other heavier products formed in the H2 + HFA reaction could not be identified. Quenching cross-sections were determined for C2H4, C3H8, C4H10, and HFA relative to that for N2O.

scholarly journals Studies of triose phosphate isomerase by hydrogen exchange

1974 ◽  
Vol 141 (3) ◽  
pp. 753-760 ◽  
Author(s):  
Christopher A. Browne ◽  
Stephen G. Waley
Keyword(s):  
Hydrogen Exchange ◽  
Marked Effect ◽  
Protein Analysis ◽  
Hollow Fibre ◽  
Triose Phosphate Isomerase ◽  
Rabbit Muscle ◽  
Structural Rearrangements ◽  
Triose Phosphate ◽  
Chicken Muscle ◽  
Low Concentrations

The3H–H exchange of chicken muscle and rabbit muscle triose phosphate isomerases was studied. Their behaviour was mostly very similar. ‘Exchange-in’ (acquisition of radioactivity when protein was incubated in3H2O) was measured at 37°C and at pH7.5, and the rates of exchange of the native and liganded enzymes were compared. Inhibitors and substrates retarded exchange, substrates showing the most marked effect; structural rearrangements in the enzyme may thus play some part in catalysis. The inhibitor phosphoglycollate affected the rabbit enzyme, but had little or no effect on the chicken enzyme. ‘Exchange-out’ (loss of radioactivity from protein previously labelled by incubation in3H2O) was measured by hollow-fibre dialysis. When ligand was removed during the course of dialysis (by replacing buffer that contained ligand with buffer that lacked ligand) there was a prompt decrease in the number of labelled H atoms of the protein. Analysis of the curves provides some information about the number and half-lives of the responsive H atoms. Ligands decrease the motility of the protein and affect about one-fifth of the chain. Low concentrations of glycerol 3-phosphate have an effect that is greater than expected.

Equilibrium Unfolding Mechanism of Chicken Muscle Triose Phosphate Isomerase

2008 ◽  
Vol 15 (4) ◽  
pp. 365-370 ◽  
Author(s):  
Yi Shi ◽  
Jiang-hong Liu ◽  
Hong-jie Zhang ◽  
Yanwei Ding
Keyword(s):  
Triose Phosphate Isomerase ◽  
Triose Phosphate ◽  
Chicken Muscle ◽  
Equilibrium Unfolding

Uniquely Labelled Active Site Sequence in Chicken Muscle Triose Phosphate Isomerase

Nature ◽  
1970 ◽  
Vol 227 (5254) ◽  
pp. 180-181 ◽  
Author(s):  
A. F. W. COULSON ◽  
J. R. KNOWLES ◽  
J. D. PRIDDLE ◽  
R. E. OFFORD
Keyword(s):  
Active Site ◽  
Triose Phosphate Isomerase ◽  
Triose Phosphate ◽  
Chicken Muscle ◽  
Active Site Sequence

Mass-spectrometric studies of ionization in flames I. The spectrometer and its application to ionization in hydrogen flames

1960 ◽  
Vol 255 (1283) ◽  
pp. 520-537 ◽  
Keyword(s):  
Nitric Oxide ◽  
Gas Phase ◽  
Reaction Zone ◽  
Heat Of Formation ◽  
The Other ◽  
Exchange Reactions ◽  
Thermal Phenomenon ◽  
Secondary Phenomenon ◽  
Kinetics Of ◽  
Positive Ion

An apparatus is described for the extraction of ions from flames burning at atmospheric pres­sure, and for the subsequent mass analysis of the ions. It consists essentially of a fine leak in a platinum or quartz diaphragm on which the flame is played, and on the other side of which a low pressure is maintained by pumping. The ions are separated by suitable disposition of electrodes in a series of expansion chambers before passing into the analyzer. It is concluded that secondary ionization is not important in the mass spectrometer, and that charge exchange reactions and other related types of reaction, in so far as they occur inside the spectrometer, largely reflect similar reactions occurring in the external flame. Ionic concentrations as low as 10 5 per cm 3 can be measured for ions ranging in mass from 10 to 400 atomic units. A brief account is given of the ionization observed from premixed flames of hydrogen, oxygen and nitrogen. The most evident positive ion was hydroxonium (H 3 O + ), the other important ones being NH + 4 and NO + . The first two of these also occurred in hydrated forms, associated with up to 4 molecules of water. These hydrates occur to a large extent in the cooler parts of the flame system (just before the reaction zone), and are considered to be a secondary phenomenon, possibly formed by association just inside the entry leak into the spectrometer. Experimental evidence is adduced for the formation of H 3 O + in the homogeneous gas phase in and near the reaction zone, rather than by catalytic interaction with the walls of the leak. The most likely reaction is considered to be H + H + OH = H 3 O + + e - and the kinetics of this are considered. Doubts about the heat of formation of this ion, and about that of the NH + 4 ion preclude quantitative decisions on many points. The ionization of nitric oxide is shown to be essentially a thermal phenomenon, by observations of ionization with known amounts of nitric oxide added to the flame gases. The relatively slow rate of recombination observed beyond the reaction zone is discussed, and found to be in line with previous results.

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