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 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.

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 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.

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.

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
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