1. The binding of L-valyl-L-tryptophan to crystalline thermolysin illustrates the mode of interaction of a product of peptide hydrolysis
H M Holden, B W Matthews J Biol Chem. 1988 Mar 5;263(7):3256-60. doi: 10.2210/pdb3tmn/pdb.
Crystallographic analysis of the binding of mercaptoacetyl-L-valyl-L-tryptophan to thermolysin suggests that this inhibitor is hydrolyzed by the crystalline enzyme. The apparent product of hydrolysis, L-valyl-L-tryptophan (Val-Trp), occupies the S1'-S2' subsites of the active site, not the S1-S1' subsites as observed previously for the dipeptide L-alanyl-L-phenylalanine (Ala-Phe). The difference in binding of Val-Trp and Ala-Phe is consistent with the specificity requirements and preferences of thermolysin. The binding of Val-Trp illustrates the mode of interaction of one of the products of peptide hydrolysis. High resolution crystallographic refinement indicates that the valyl amino group makes three hydrogen bonds to the enzyme and to solvent and, in addition, is 2.8 A from the carboxylate of Glu-143. This is the first instance in which a direct interaction has been observed between Glu-143 and the scissile nitrogen. As such, the study directly supports the mechanism of action for thermolysin proposed by Hangauer et al. (Hangauer, D. G., Monzingo, A. F., and Matthews, B. W. (1984) Biochemistry 23, 5730-5741) and, by analogy, indirectly supports the similar mechanism proposed for carboxypeptidase A (Monzingo, A. F., and Matthews, B. W. (1984) Biochemistry 23, 5724-5729).
3. L-tryptophan 2',3'-oxidase from Chromobacterium violaceum. Substrate specificity and mechanistic implications
R Genet, P H Bénetti, A Hammadi, A Ménez J Biol Chem. 1995 Oct 6;270(40):23540-5. doi: 10.1074/jbc.270.40.23540.
L-Tryptophan 2',3'-oxidase, an amino acid alpha,beta-dehydrogenase isolated from Chromobacterium violaceum, catalyzes the formation of a double bond between the C alpha and C beta carbons of various tryptophan derivatives provided that they possess: (i) a L-enantiomeric configuration, (ii) an alpha-carbonyl group, and (iii) an unsubstituted and unmodified indole nucleus. Kinetic parameters were evaluated for a series of tryptophan analogues, providing information on the contribution of each chemical group to substrate binding. The stereochemistry of the dehydro product was determined to be a Z-configuration from proton nuclear magnetic resonance assignments. No reaction can be observed in the presence of other aromatic beta-substituted alanyl residues which behave neither as substrates nor as inhibitors and therefore do not compete against this reaction. The enzymatic synthesis of alpha,beta-dehydrotryptophanyl peptides from 5 to 24 residues was successfully achieved without side product formation, irrespective of the position of the tryptophan residue in the amino acid sequence. A reactional mechanism involving a direct alpha,beta-dehydrogenation of the tryptophan side chain is proposed.
