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.

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 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.

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

scholarly journals The existence of an electrophilic component in the reaction catalysed by triose phosphate isomerase

1974 ◽  
Vol 141 (2) ◽  
pp. 589-592 ◽  
Author(s):  
Martin R. Webb ◽  
Jeremy R. Knowles
Keyword(s):  
Carbonyl Group ◽  
Triose Phosphate Isomerase ◽  
Dihydroxyacetone Phosphate ◽  
Free Solution ◽  
Triose Phosphate ◽  
Order Of Magnitude

In the presence of triose phosphate isomerase, the substrate dihydroxyacetone phosphate is reduced stereoselectively by NaBH4. The reduction of enzyme-bound substrate is almost completely or completely stereoselective and occurs about one order of magnitude faster than that in free solution. This acceleration implies a polarization of the carbonyl group when dihydroxyacetone phosphate is bound.

scholarly journals Loss of Peroxisomal Hydroxypyruvate Reductase Inhibits Triose Phosphate Isomerase but Stimulates Cyclic Photosynthetic Electron Flow and the Glc-6P-Phosphate Shunt

2018 ◽  
Author(s):  
Jiying Li ◽  
Sarathi M. Weraduwage ◽  
Alyssa L. Preiser ◽  
Sean E. Weise ◽  
Deserah D. Strand ◽  
...  
Keyword(s):  
Photosystem I ◽  
Electron Flow ◽  
Competitive Inhibitor ◽  
Triose Phosphate Isomerase ◽  
Cyclic Electron Flow ◽  
Dihydroxyacetone Phosphate ◽  
Triose Phosphate ◽  
Hydroxypyruvate Reductase ◽  
Photosynthetic Electron Flow ◽  
Mutant Lines

AbstractThe oxygenation of ribulose 1,5-bisphosphate by Rubisco is the first step in photorespiration and reduces the efficiency of photosynthesis in C3 plants. Our recent data indicates that mutants in photorespiration have increased rates of photosynthetic cyclic electron flow around photosystem I. We investigated mutant lines lacking peroxisomal hydroxypyruvate reductase to determine if there are connections between 2-PG accumulation and cyclic electron flow. We found that 2-PG is a competitive inhibitor of triose phosphate isomerase (TPI), an enzyme in the Calvin-Benson cycle that converts glyceraldehyde 3-phosphate to dihydroxyacetone phosphate. This block in metabolism could be overcome if glyceraldehyde 3-phosphate is exported to the cytosol where the cytosolic triose phosphate isomerase could convert it to dihydroxyacetone phosphate. We found evidence that carbon is reimported as Glc-6P-phosphate forming a cytosolic bypass around the block of stromal TPI. However, this also stimulates a Glc-6P-phosphate shunt, which consumes ATP, which can be compensated by higher rates of cyclic electron flow.Once Sentence SummaryTriose phosphate isomerase is inhibited in plants lacking hydroxypyruvate reductase 1 and this is overcome by exporting triose phosphate to the cytosol and importing Glc-6P, which stimulates a Glc-6P-phosphate shunt and cyclic electron flow.

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