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Evolutionary specialization of a tryptophan indole group for transition-state stabilization by eukaryotic transglutaminases

Iismaa, Siiri E., Holman, Sara, Wouters, Merridee A., Lorand, Laszlo, Graham, Robert M. and Husain, Ahsan 2003, Evolutionary specialization of a tryptophan indole group for transition-state stabilization by eukaryotic transglutaminases, Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 22, pp. 12636-12641, doi: 10.1073/pnas.1635052100.

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Title Evolutionary specialization of a tryptophan indole group for transition-state stabilization by eukaryotic transglutaminases
Author(s) Iismaa, Siiri E.
Holman, Sara
Wouters, Merridee A.
Lorand, Laszlo
Graham, Robert M.
Husain, Ahsan
Journal name Proceedings of the National Academy of Sciences of the United States of America
Volume number 100
Issue number 22
Start page 12636
End page 12641
Total pages 6
Publisher National Academy of Sciences
Place of publication Washington, D.C.
Publication date 2003-10-28
ISSN 0027-8424
1091-6490
Keyword(s) acylation
amino acid substitution
article
catalysis
chemical reaction kinetics
cross linking
deacylation
ecological specialization
enzyme active site
enzyme kinetics
eukaryote
evolutionary adaptation
Michaelis constant
protein modification
Summary Covalent posttranslational protein modifications by eukaryotic transglutaminases proceed by a kinetic pathway of acylation and deacylation. Ammonia is released as the acylenzyme is formed, whereas the cross-linked product is released later in the deacylation step. Superposition of the active sites of transglutaminase type 2 (TG2) and the structurally related cysteine protease, papain, indicates that in the formation of tetrahedral intermediates, the backbone nitrogen of the catalytic Cys-277 and the NƐ1 nitrogen of Trp-241 of TG2 could contribute to transition-state stabilization. The importance of this Trp-241 side chain was demonstrated by examining the kinetics of dansylcadaverine incorporation into a model peptide. Although substitution of the Trp-241 side chain with Ala or Gly had only a small effect on the Michaelis constant Km (1.5-fold increase), it caused a >300-fold lowering of the catalytic rate constant kcat. The wild-type and mutant TG2-catalyzed release of ammonia showed kinetics similar to the kinetics for the formation of cross-linked product, indicating that transitionstate stabilization in the acylation step was rate-limiting. In papain, a Gln residue is at the position of TG2-Trp-241. The conservation of Trp-241 in all eukaryotic transglutaminases and the finding that W241Q-TG2 had a much lower kcat than wild-type enzyme suggest evolutionary specialization in the use of the indole group. This notion is further supported by the observation that transitionstate- stabilizing side chains of Tyr and His that operate in some serine and metalloproteases only partially substituted for Trp.
Language eng
DOI 10.1073/pnas.1635052100
Field of Research 060199 Biochemistry and Cell Biology not elsewhere classified
Socio Economic Objective 970106 Expanding Knowledge in the Biological Sciences
HERDC Research category C1.1 Refereed article in a scholarly journal
Copyright notice ©2003, National Academy of Sciences
Persistent URL http://hdl.handle.net/10536/DRO/DU:30038981

Document type: Journal Article
Collection: School of Life and Environmental Sciences
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