Histidine modification with

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. 

Natural abundance C n.m.r. spectroscopy has been used to study the carboxymethylation of L-histidine-15 of hen egg white lysozyme upon treatment with iodoacetate. A comparison of the spectrum of chromatographically pure (A -carboxymethyl-L-histidine) lysozyme with that of the intact protein indicated that the chemical modification did not significantly affect the conformation of the protein. 

A final example implies no surface modification. Using magnetite nanoparticles as seeds, Sun et al. [164] described the synthesis of magnetic core-poly(AAm) shell particles obtained by UV irradiation of an aqueous solution of F03O4, AAm, and MBA. The surface of the particles was then modified to introduce amino groups, subsequently linked to L-histidine labeled with Re, one of the most efficient radioisotopes for cancer radiotherapy. 

MnClj typically inhibits DPC supported DCIP photoreduction of PS II membranes by 50% under the conditions of our assays (Fig. 1). Modification of available histidine residues with the modifying agent DEPC reduces the inhibition by MnClj to 25% [4,5]. Modification of carboxyl residues with EDC results in a similar reduction. Sequential modification of residues, first with DEPC and then with EDC results in MnCl2 being unable to inhibit DPC -> DCIP activity. Half of the high-affinity Mn-binding site is susceptible to DEPC and the other half to EDC. 

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...

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... 

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. 

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