Functional role of the conserved active site proline of triosephosphate isomerase

Marco G. Casteleijn, Markus Alahuhta, Katrin Groebel, Ibrahim El-Sayed, Koen Augustyns, Anne Marie Lambeir, Peter Neubauer, Rik K. Wierenga

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Abstract

The importance of the fully conserved active site proline, Pro168, for the reaction mechanism of triosephosphate isomerase (TIM) has been investigated by studying the enzymatic and crystallographic properties of the P168A variant of trypanosomal TIM. In TIM, Pro168 follows the key catalytic residue Glu167, situated at the beginning of the flexible active site loop (loop 6). Turnover numbers of the P168A variant for its substrates are reduced approximately 50-fold, whereas the Km values are approximately 2 times lower. The affinity of the P168A variant for the transition state analogue 2-phosphoglycolate (2PG) is reduced 5-fold. The crystal structures of unliganded and liganded (2PG) P168A show that the phosphate moiety of 2PG is bound similarly as in wild-type TIM, whereas the interactions of the carboxylic acid moiety with the side chain of the catalytic Glu 167 differ. The unique properties of the proline side chain at position 168 are required to transmit ligand binding to the conformational change of Glu167: the side chain of Glu 167 flips from the inactive swung-out to the active swung-in conformation on ligand binding in wild-type TIM, whereas in the mutant this conformational change does not occur. Further structural comparisons show that in the wild-type enzyme the concerted movement of loop 6 and loop 7 from unliganded-open to liganded-closed appears to be facilitated by the interactions of the phosphate moiety with loop 7. Apparently, the rotation of 90° of the Gly211-Gly212 peptide plane of loop 7 plays a key role in this concerted movement.
Original languageEnglish
Pages (from-to)15483-15494
Number of pages12
JournalBiochemistry
Volume45
Issue number51
DOIs
Publication statusPublished - 26 Dec 2006
MoE publication typeA1 Journal article-refereed

Fingerprint

Triose-Phosphate Isomerase
Peptidylprolyl Isomerase
Proline
Catalytic Domain
Phosphates
Ligands
Carboxylic Acids
Conformations
Crystal structure
Peptides
Substrates
Enzymes
phosphoglycolate

Keywords

  • 2-phosphoglycolate (2PG)
  • crystallographic properti
  • Triosephosphate isomerase (TIM
  • phosphate moiety

Cite this

Casteleijn, M. G., Alahuhta, M., Groebel, K., El-Sayed, I., Augustyns, K., Lambeir, A. M., ... Wierenga, R. K. (2006). Functional role of the conserved active site proline of triosephosphate isomerase. Biochemistry, 45(51), 15483-15494. https://doi.org/10.1021/bi061683j
Casteleijn, Marco G. ; Alahuhta, Markus ; Groebel, Katrin ; El-Sayed, Ibrahim ; Augustyns, Koen ; Lambeir, Anne Marie ; Neubauer, Peter ; Wierenga, Rik K. / Functional role of the conserved active site proline of triosephosphate isomerase. In: Biochemistry. 2006 ; Vol. 45, No. 51. pp. 15483-15494.
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abstract = "The importance of the fully conserved active site proline, Pro168, for the reaction mechanism of triosephosphate isomerase (TIM) has been investigated by studying the enzymatic and crystallographic properties of the P168A variant of trypanosomal TIM. In TIM, Pro168 follows the key catalytic residue Glu167, situated at the beginning of the flexible active site loop (loop 6). Turnover numbers of the P168A variant for its substrates are reduced approximately 50-fold, whereas the Km values are approximately 2 times lower. The affinity of the P168A variant for the transition state analogue 2-phosphoglycolate (2PG) is reduced 5-fold. The crystal structures of unliganded and liganded (2PG) P168A show that the phosphate moiety of 2PG is bound similarly as in wild-type TIM, whereas the interactions of the carboxylic acid moiety with the side chain of the catalytic Glu 167 differ. The unique properties of the proline side chain at position 168 are required to transmit ligand binding to the conformational change of Glu167: the side chain of Glu 167 flips from the inactive swung-out to the active swung-in conformation on ligand binding in wild-type TIM, whereas in the mutant this conformational change does not occur. Further structural comparisons show that in the wild-type enzyme the concerted movement of loop 6 and loop 7 from unliganded-open to liganded-closed appears to be facilitated by the interactions of the phosphate moiety with loop 7. Apparently, the rotation of 90° of the Gly211-Gly212 peptide plane of loop 7 plays a key role in this concerted movement.",
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Casteleijn, MG, Alahuhta, M, Groebel, K, El-Sayed, I, Augustyns, K, Lambeir, AM, Neubauer, P & Wierenga, RK 2006, 'Functional role of the conserved active site proline of triosephosphate isomerase', Biochemistry, vol. 45, no. 51, pp. 15483-15494. https://doi.org/10.1021/bi061683j

Functional role of the conserved active site proline of triosephosphate isomerase. / Casteleijn, Marco G.; Alahuhta, Markus; Groebel, Katrin; El-Sayed, Ibrahim; Augustyns, Koen; Lambeir, Anne Marie; Neubauer, Peter; Wierenga, Rik K.

In: Biochemistry, Vol. 45, No. 51, 26.12.2006, p. 15483-15494.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Functional role of the conserved active site proline of triosephosphate isomerase

AU - Casteleijn, Marco G.

AU - Alahuhta, Markus

AU - Groebel, Katrin

AU - El-Sayed, Ibrahim

AU - Augustyns, Koen

AU - Lambeir, Anne Marie

AU - Neubauer, Peter

AU - Wierenga, Rik K.

PY - 2006/12/26

Y1 - 2006/12/26

N2 - The importance of the fully conserved active site proline, Pro168, for the reaction mechanism of triosephosphate isomerase (TIM) has been investigated by studying the enzymatic and crystallographic properties of the P168A variant of trypanosomal TIM. In TIM, Pro168 follows the key catalytic residue Glu167, situated at the beginning of the flexible active site loop (loop 6). Turnover numbers of the P168A variant for its substrates are reduced approximately 50-fold, whereas the Km values are approximately 2 times lower. The affinity of the P168A variant for the transition state analogue 2-phosphoglycolate (2PG) is reduced 5-fold. The crystal structures of unliganded and liganded (2PG) P168A show that the phosphate moiety of 2PG is bound similarly as in wild-type TIM, whereas the interactions of the carboxylic acid moiety with the side chain of the catalytic Glu 167 differ. The unique properties of the proline side chain at position 168 are required to transmit ligand binding to the conformational change of Glu167: the side chain of Glu 167 flips from the inactive swung-out to the active swung-in conformation on ligand binding in wild-type TIM, whereas in the mutant this conformational change does not occur. Further structural comparisons show that in the wild-type enzyme the concerted movement of loop 6 and loop 7 from unliganded-open to liganded-closed appears to be facilitated by the interactions of the phosphate moiety with loop 7. Apparently, the rotation of 90° of the Gly211-Gly212 peptide plane of loop 7 plays a key role in this concerted movement.

AB - The importance of the fully conserved active site proline, Pro168, for the reaction mechanism of triosephosphate isomerase (TIM) has been investigated by studying the enzymatic and crystallographic properties of the P168A variant of trypanosomal TIM. In TIM, Pro168 follows the key catalytic residue Glu167, situated at the beginning of the flexible active site loop (loop 6). Turnover numbers of the P168A variant for its substrates are reduced approximately 50-fold, whereas the Km values are approximately 2 times lower. The affinity of the P168A variant for the transition state analogue 2-phosphoglycolate (2PG) is reduced 5-fold. The crystal structures of unliganded and liganded (2PG) P168A show that the phosphate moiety of 2PG is bound similarly as in wild-type TIM, whereas the interactions of the carboxylic acid moiety with the side chain of the catalytic Glu 167 differ. The unique properties of the proline side chain at position 168 are required to transmit ligand binding to the conformational change of Glu167: the side chain of Glu 167 flips from the inactive swung-out to the active swung-in conformation on ligand binding in wild-type TIM, whereas in the mutant this conformational change does not occur. Further structural comparisons show that in the wild-type enzyme the concerted movement of loop 6 and loop 7 from unliganded-open to liganded-closed appears to be facilitated by the interactions of the phosphate moiety with loop 7. Apparently, the rotation of 90° of the Gly211-Gly212 peptide plane of loop 7 plays a key role in this concerted movement.

KW - 2-phosphoglycolate (2PG)

KW - crystallographic properti

KW - Triosephosphate isomerase (TIM

KW - phosphate moiety

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U2 - 10.1021/bi061683j

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Casteleijn MG, Alahuhta M, Groebel K, El-Sayed I, Augustyns K, Lambeir AM et al. Functional role of the conserved active site proline of triosephosphate isomerase. Biochemistry. 2006 Dec 26;45(51):15483-15494. https://doi.org/10.1021/bi061683j