Histidine modification

Ohmiya, Y., and Tsuji, F. I. (1993). Bioluminescence of the Ca2+-binding photoprotein aequorin, after histidine modification. FEBS Lett. 320 267-270. 

In the catalysis of the lyase from C. perfringens, the participation of lysine residues forming intennediary Schiff bases between enzyme and substrate molecules, and of histidine residues, has been demonstrated with the aid of photooxidation, reagents for histidine modification, and borohydride reduction in the presence of substrate.408-418 Thus, according to Frazi and coworkers,414 the lyase belongs to the class I lyases (aldolases). The catalytic mechanism proposed is outlined in Scheme 3. Evidence has been educed for the existence of a similar mechanism of cleavage of sialic acid by the lyase enriched from pig kidney.411... 

When RNase-S was treated with iodoacetate at pH 6, both inactivation and histidine modification occurred 164). The modified histidine was in S-protein and was assumed to be His 119 since the sole product on analysis was 1-CM-His. In the absence of S-peptide only methionine modification occurred in S-protein. The loss of potential activity probably resulted from the reaction of the second of the two modifiable Met residues. The location of these residues in the sequence was not established. 

Fig. 1. Correlation between inhibition of the RrFi-ATPase and the modification of histidyl residues by DEPC or the binding of [I ClDCCD and [1 C]NBD-C1 to the RrFi. RrFi mg/ml) was inactivated by 250 yM DEPC added at 10 min intervals in portions of 50 yM. After each addition the Ca +-ATP activity and histidine modification were measured as described by Khananshvili, Gromet-Elhanan (1983c). RrFi (1 mg/ml) was incubated with 100 UM [I cIdCCD or 300 uM [14c]NBD-C1 for various time in-tervals, freed from untreated reagent and assayed for activity and binding as described...

To date, only two exceptions to the pK of 8 rule have been found the Rieske protein from Sulfolobus acidocaldarius (139) and that from Thiobacillus ferrooxidans (140). In both cases, a first pK is observed in the vicinity of 6 (Fig. 7). The fact that Sulfolobus and Thiobacillus are phylogenetically almost as distant as they can possibly be, but share acidophilic growth conditions (medium-pH of 2), indicates that the pK, which is lower by 2 pH units in Sulfolobus and Thiobacillus, reflects adaptation. In the absence of structural information for the two acidophilic Rieske proteins, the molecular modifications resulting in this pK shift are difficult to guess. The absence of sequence data for the Thiobacillus protein furthermore precludes a comparative approach. It seems likely, however, that the solvent-exposed histidine ligands to the cluster will become slightly more bur-... 

There is only one tyrosine residue in some sea snake neurotoxins. This residue is usually quite difficult to modify, but once it is modified, the toxicity is lost (9). Histidine seems not to be essential as the chemical modification of this residue does not affect the toxicity 10). 

Similarly, the rate of inhibition of phosphoenzyme formation by diethylpyrocarbonate (DEPC) was much slower than the loss of ATPase activity [368], Even when the reaction approached completion with more than 90% inhibition of ATP hydrolysis, about 70% of the Ca -ATPase could still be phosphorylated by ATP (2.3nmoles of E P/mg protein). The remaining 30% of E P formation and the corresponding ATPase activity was not reactivated by hydroxylamine treatment, suggesting some side reaction with other amino acids, presumably lysine. When the reaction of the DEPC-modified ATPase with P-ATP was quenched by histidine buffer (pH 7.8) the P-phosphoenzyme was found to be exceptionally stable under the same conditions where the phosphoenzyme formed by the native ATPase underwent rapid hydrolysis [368]. The nearly normal phosphorylation of the DEPC-trea-ted enzyme by P-ATP implies that the ATP binding site is not affected by the modification, and the inhibition of ATPase activity is due to inhibition of the hydrolysis of the phosphoenzyme intermediate [368]. This is in contrast to an earlier report by Tenu et al. [367], that attributed the inhibition of ATPase activity by... 

Chemical modifications of proteins (enzymes) by reacting them with iV-acylimidazoles are a way of studying active sites. By this means the amino acid residues (e.g., tyrosine, lysine, histidine) essential for catalytic activity are established on the basis of acylation with the azolides and deacylation with other appropriate reagents (e.g., hydroxylamine). 

The introduction of redox activity through a Co11 center in place of redox-inactive Zn11 can be revealing. Carboxypeptidase B (another Zn enzyme) and its Co-substituted derivative were oxidized by the active-site-selective m-chloroperbenzoic acid.1209 In the Co-substituted oxidized (Co111) enzyme there was a decrease in both the peptidase and the esterase activities, whereas in the zinc enzyme only the peptidase activity decreased. Oxidation of the native enzyme resulted in modification of a methionine residue instead. These studies indicate that the two metal ions impose different structural and functional properties on the active site, leading to differing reactivities of specific amino acid residues. Replacement of zinc(II) in the methyltransferase enzyme MT2-A by cobalt(II) yields an enzyme with enhanced activity, where spectroscopy also indicates coordination by two thiolates and two histidines, supported by EXAFS analysis of the zinc coordination sphere.1210... 

Many recent studies have focused on the mechanisms of formaldehyde modification, cross-linking, and reversal.19,37 8 In general, these studies found that formaldehyde is very specific, particularly when reaction times are relatively short. The amino-termini, lysine, tryptophan, and cysteine are the targets of modification in this case. Longer reaction times reveal more extensive modifications, including arginine, histidine, tyrosine, and phenylalanine. 

The most significant amino acids for modification and conjugation purposes are the ones containing ionizable side chains aspartic acid, glutamic acid, lysine, arginine, cysteine, histidine, and tyrosine (Figure 1.6). In their unprotonated state, each of these side chains can be potent nucleophiles to engage in addition reactions (see the discussion on nucleophilicity below). 

Figure 2.5 The cisplatin reactive group can covalently couple to methionine-, cysteine-, and histidine-containing peptides or proteins. It also reacts with guanine groups to form a covalent modification on the N7 nitrogen.

The addition of a radioactive iodine atom to a protein molecule typically has little effect on the resultant protein activity, unless the active center is modified in the process. The size of an iodine atom is relatively small and does not result in many steric problems with large molecules. The sites of potential protein modification are tyrosine and histidine side chains. Tyrosine may be modified with a total of two iodine atoms per phenolate group, whereas histidine can incorporate one iodine. Sulfhydryl modification at cysteine residues is typically unstable. 

Directing the iodination reaction toward histidine residues in proteins, as opposed to principally tyrosine modification, is possible simply by increasing the pH of the lodobeads reaction from the manufacturer s recommended pH 7.0-8.2 (Tsomides et ai, 1991). No reducing agent is required to stop the iodination reaction as is the case with chloramine-T and other methods. 

---