Triosephosphate Isomerase: Isotope Studies on the Mechanistic Pathway

Knowles, Jeremy R.; Leadlay, Peter F.; Maister, Selwyn G.

doi:10.1101/sqb.1972.036.01.022

Cited by 17 publications

(7 citation statements)

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“…If the group with pK 6 had to be protonated and that with pK 9 had to be ionized, only 0.1% of the enzyme would be correctly protonated at p H 7.5 and V / K E t could not be larger than times the diffusion-limiting rate. With glyceraldehyde-3-P, however, VIKE, is nearly 3 x 10' M -' s e~' (70), and thus this hypothesis is not possible.…”

Section: Reverse Protonatwnmentioning

confidence: 94%

“…This exchange is so rapid that essentially no label from dihydroxyacetone-P-2-T is found in glyceraldehyde-P after reaction, and there is no isotope effect in the other direction with glyceraldehyde-P-1 -D because the label exchanges prior to the rate-limiting step, which is conversion of the enediol to dihydroxyacetone-P (70). Possibly Cys-126 or some of the other polar groups at the bottom of the active site facilitate proton exchange with the solvent through the back side of the protein; it is hard to imagine that the exchange takes place through the mouth of the active site.…”

Section: Tkiose-p Isomerasementioning

confidence: 99%

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Determining the Chemical Mechanisms of Enzyme‐Catalyzed Reactions by Kinetic Studies

Cleland¹

1977

Advances in Enzymology - And Related Areas of Molecular Biology

194

129

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Section: Reverse Protonatwnmentioning

confidence: 94%

Section: Tkiose-p Isomerasementioning

confidence: 99%

Determining the Chemical Mechanisms of Enzyme‐Catalyzed Reactions by Kinetic Studies

Cleland¹

1977

Advances in Enzymology - And Related Areas of Molecular Biology

194

129

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“…Triose phosphate isomerase (EC 5.3.1.1) was until recently little studied. However, the superficial simplicity of the elementary reactions ofcatalysis and its small size among glycolytic enzymes has now led to efforts in many laboratories to elucidate the catalytic mechanism and covalent structure (for reviews of this work see Banner et al, 1971;Knowles et al, 1971).…”

mentioning

confidence: 99%

Studies on the subunit structure and amino acid sequence of triose phosphate isomerase from chicken breast muscle

Furth

Milman

Priddle

et al. 1974

Biochemical Journal

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1. Triose phosphate isomerase was prepared by chromatography on DEAE-cellulose of an (NH(4))(2)SO(4) fraction of an extract of homogenized chicken breast muscle. The product is homogeneous on gel electrophoresis and is suitable for growing crystals for X-ray work. The specific activity is 10000 units/mg and the value for E(0.1%) (280) is 1.20. 2. Comparison between the sum of the amino acid compositions of the tryptic peptides of the protein and the amino acid composition obtained on total hydrolysis of the protein indicates that the relative subunit mass is about 27000. 3. These data, together with the results of the examination of the amino acid compositions of a number of minor peptides, the number of peptides in the tryptic digest and the complete amino acid sequences of the tryptic peptides (the determination of which is described here), give no indication that the subunits are dissimilar. 4. A tentative amino acid sequence is presented for the protein, in which the ordering of the tryptic peptides is derived by homology with the sequence of the rabbit muscle enzyme (Corran & Waley, 1973). 5. An appendix describes the use that was made of mass spectrometry in the determination of some of the sequences. Mass-spectrometric data have been obtained for 35 residues, that is about 15% of the total sequence of the protein. 6. An extended version of the present paper has been deposited as Supplementary Publication SUP 50025 at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.

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“…The high specificity ofbromohydroxyacetone phosphate for triose phosphate isomerase can be turned to good account, however. The isomerase is a remarkably effective catalyst (kcat./Km for glyceraldehyde 3-phosphate is 2 x 108M-1 * S-1; Knowles et aL., 1971) and a very small contamination of other glycolytic enzymes by isomerase can, on a weight basis, still result in unacceptably high isomerase activities. Many of the experiments reported elsewhere (Knowles et al, 1971) required isomerase-free preparations of aldolase, glyceraldehyde 3-phosphate dehydrogenase and a-glycerophosphate dehydrogenase.…”

Section: Discussionmentioning

confidence: 99%

“…The isomerase is a remarkably effective catalyst (kcat./Km for glyceraldehyde 3-phosphate is 2 x 108M-1 * S-1; Knowles et aL., 1971) and a very small contamination of other glycolytic enzymes by isomerase can, on a weight basis, still result in unacceptably high isomerase activities. Many of the experiments reported elsewhere (Knowles et al, 1971) required isomerase-free preparations of aldolase, glyceraldehyde 3-phosphate dehydrogenase and a-glycerophosphate dehydrogenase. Brief (10min) incubation of these enzymes at pH7 with 10 lM-bromohydroxyacetone phosphate, followed by gel filtration, allows the isolation of enzymes containing undetectably low isomerase activity.…”

Section: Discussionmentioning

confidence: 99%

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

Mare

Coulson

Knowles

et al. 1972

Biochemical Journal

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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.

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Triosephosphate Isomerase: Isotope Studies on the Mechanistic Pathway

Cited by 17 publications

References 0 publications

Determining the Chemical Mechanisms of Enzyme‐Catalyzed Reactions by Kinetic Studies

Determining the Chemical Mechanisms of Enzyme‐Catalyzed Reactions by Kinetic Studies

Studies on the subunit structure and amino acid sequence of triose phosphate isomerase from chicken breast muscle

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

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