title: Evolutionary specialization of a tryptophan indole group for transition-state stabilization by eukaryotic transglutaminases.
abstract: 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 N1 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 transition-state 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 transition-state-stabilizing side chains of Tyr and His that operate in some serine and metalloproteases only partially substituted for Trp.
authors.affiliation: Victor Chang Cardiac Research Institute, 384 Victoria Street, Darlinghurst, NSW 2010, Australia. s.iismaa@victorchang.unsw.edu.au
authors.forename: Siiri E
authors.initials: SE
authors.lastname: Iismaa
authors.forename: Sara
authors.initials: S
authors.lastname: Holman
authors.forename: Merridee A
authors.initials: MA
authors.lastname: Wouters
authors.forename: Laszlo
authors.initials: L
authors.lastname: Lorand
authors.forename: Robert M
authors.initials: RM
authors.lastname: Graham
authors.forename: Ahsan
authors.initials: A
authors.lastname: Husain
mesh_terms: D000215:Acylation; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004795:Enzyme Stability; D019143:Evolution, Molecular; D019204:GTP-Binding Proteins / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007211:Indoles; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D011503:Transglutaminases / Q000737:chemistry*; D014364:Tryptophan
chemical_list: D000345:Affinity Labels; D007211:Indoles; D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; D014364:Tryptophan; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins