scholarly journals Active-site labelling of triose phosphate isomerase. The reaction of bromohydroxyacetone phosphate with a unique glutamic acid residue and the migration of the label to tyrosine

1972 ◽  
Vol 129 (2) ◽  
pp. 321-331 ◽  
Author(s):  
Susan De La Mare ◽  
A. F. W. Coulson ◽  
J. R. Knowles ◽  
J. D. Priddle ◽  
R. E. Offord
Keyword(s):  
Glutamic Acid ◽  
Active Site ◽  
Triose Phosphate Isomerase ◽  
Primary Site ◽  
Primary Proton ◽  
Carboxylate Group ◽  
Irreversible Inhibitor ◽  
Triose Phosphate ◽  
Glutamic Acid Residue ◽  
Chicken Muscle

Triose phosphate isomerase from chicken muscle reacts stoicheiometrically with the active-site-directed irreversible inhibitor bromohydroxyacetone phosphate with concomitant loss of all catalytic activity. The primary site of attachment has been shown to be a unique glutamic acid residue in the sequence Ala-Tyr-Glu-Pro-Val-Trp. Unless the inhibitor–enzyme bond is stabilized by reduction of the C-2 carbonyl group with borohydride, the phosphate group is lost and the label migrates to the adjacent tyrosine residue. It is suggested that the γ-carboxylate group of the glutamic acid residue may be the base responsible for primary proton abstraction from substrate in the catalysis. The failure of this reagent specifically to inactivate either muscle or yeast aldolase, and the use of the reagent in preparing isomerase-free glycolytic enzymes, is discussed.

scholarly journals The active centre of rabbit muscle triose phosphate isomerase. The site that is labelled by glycidol phosphate

1971 ◽  
Vol 123 (2) ◽  
pp. 163-170 ◽  
Author(s):  
Janet C. Miller ◽  
S. G. Waley
Keyword(s):  
Phosphoric Acid ◽  
Glutamic Acid ◽  
Inorganic Phosphate ◽  
Triose Phosphate Isomerase ◽  
Rabbit Muscle ◽  
Irreversible Inhibitor ◽  
Active Centre ◽  
Triose Phosphate ◽  
Glutamic Acid Residue

1. Glycidol (2,3-epoxypropanol) phosphate is a specific irreversible inhibitor of rabbit muscle triose phosphate isomerase (EC 5.3.1.1); the site of attachment has now been studied. 2. The labelled enzyme was digested with pepsin and a modified peptide isolated. The sequence of the peptide is: Ala-Tyr-Glu-Pro-Val-Trp. 3. It is the glutamic acid residue in this peptide that is labelled: the peptide is thus a γ-glutamyl ester derived from glycerol phosphoric acid. The same site is labelled by a mixture of glycidol and inorganic phosphate. 4. Kinetic and stereochemical features of these reactions are discussed.

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

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.

scholarly journals The Identification of a Glutamic Acid Residue as Part of the Active Site of Ribonuclease T1

1967 ◽  
Vol 242 (20) ◽  
pp. 4682-4690 ◽  
Author(s):  
Kenji Takahashi ◽  
William H. Stein ◽  
Stanford Moore
Keyword(s):  
Glutamic Acid ◽  
Active Site ◽  
Ribonuclease T1 ◽  
Glutamic Acid Residue

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 The ionization of a buried glutamic acid is thermodynamically linked to the stability of Leishmania mexicana triose phosphate isomerase

2000 ◽  
Vol 267 (9) ◽  
pp. 2516-2524 ◽  
Author(s):  
Anne-Marie Lambeir ◽  
Jan Backmann ◽  
Javier Ruiz-Sanz ◽  
Vladimir Filimonov ◽  
Jens Erik Nielsen ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Triose Phosphate Isomerase ◽  
Leishmania Mexicana ◽  
Triose Phosphate ◽  
The Stability

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.

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