chapter  14
44 Pages

Modication of Histidine

A substantial number of the studies on the modication of histidine have been directed at the importance of this amino acid residue in the catalytic mechanism of enzymes and somewhat less in the function of protein binding sites. Thus, many of the modifying reagents have been directed at the enzyme active site and are considered to be afnity labels33 such as tosyl-phenylalanine chloromethyl ketone34 or 2′(3′)-O-bromoacetyluridine.35 TPCK reacts with active-site histidine (His57) in chymotrypsin, while 2′(3′)-O-bromoacetyluridine reacts with His12 in bovine pancreatic ribonuclease. The reaction rate of 2′(3′)-O-bromoacetyluridine with RNAse is approximately 3000 times than with free histidine, and the rate of inactivation of RNAse is 4.5 times faster than that observed with bromoacetate. The development of 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK, N-α-p-tosyl-llysine chloromethyl ketone)36,37 followed the development of TPCK by Elliott Shaw and coworkers.34 It is my understanding that the synthesis of the TLCK presented somewhat more of a challenge than TPCK. This material has the somewhat interesting characteristic of smelling like stale popcorn. The rate of trypsin inactivation by TLCK at pH 7.0 was determined to be 12.6 M-1 s-1, the rate of inactivation by L-lysine chloromethyl ketone was determined to be 6.1 M-1 s-1, and D-TLCK is inactive.37 There is some other work that occurred at the same time as the elegant work of Shaw and coworkers, which deserves mention. Inagami38 observed that iodoacetamide was a poor inhibitor of trypsin (3.3 × 10-2 M-1 min-1 at pH 7.0); the inclusion of methylguanidine increased the rate inactivation to 2 × 10-1 M-1 s-1. It would seem that binding of the methylguanidine changes the conformation of trypsin increasing reactivity of the active-site histidine. This is supported by the work showing a conformational change in trypsin on the binding of Nα-benzoyl-l-arginine ethyl ester.39 It would be predicted that the rate of reaction of chloroacetamide with the active-site histidine in trypsin would

FIGURE 14.1 Structure of histidine and some derivative forms. Shown is the structure of histidine, imidazole, and histamine. In the following is a schematic of the interaction of a hexahistidine tag with a nitrilotriacetic acid matrix (Adapted from Hellman, L.M., Zhao, C., Melikishvili, M. et al., Histidine-tag-directed chromophores for tracer analyses in the analytical ultracentrifuge, Methods 54, 31-38, 2011). Shown at the bottom is a schematic drawing of imidazole SAM for the metal-ion immobilization of histidine-tagged peptides (Zaltouna, A.J. and Lai, R.Y., Design and characterization of a metal ion-imidazole self-assembled monolayer for reversible immobilization of histidine-tagged peptides, Chem. Commun. 47, 12391-12393, 2011).