Tyrosine modification with

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. 

The catalytically essential nature of tyrosine 85 and its proximity to the substrate binding site and to tyrosine 115 were demonstrated from studies of modification with tetranitromethane (71) and from studies of intramolecular cross-linking of aminotyrosyl residues (72). The bro-moacetamidophenyl (69) and diazonium (70) reagents obtained from aminophenyl-pdT both react selectively and exclusively with tyrosine 85. This residue is situated, stereochemically, such that its hydroxyl group can interact with the 3 -phosphate of pdTp. 

Kievan and Tse (1983) have examined the effect of chemical modification of E. coli topoisomerase I and DNA gyrase by tetranitromethane, which reacts preferentially with tyrosine residues. With each enzyme, treatment with tetranitromethane led to abolition of the topoisomerase activity. Moreover, the enzymes were protected from this inactivation when bound to DNA, implying that some of the modified residues are involved in DNA binding. However, this study does not identify the tyrosine residues involved in the protein-DNA bond as being the amino acid residues whose modification inactivates the enzyme. In the case of DNA gyrase, which has two subunits, it was not determined which subunit was inactivated. 

Circular dichroism (CD) has played an important role in our studies on the modification of enzymes and hormones with Co(III). The objective of these studies has been to incorporate selectively substitution inert metal ions at specifically modified sites in proteins as probes of biological function. Significant information concerning the catalytic mechanism of carboxypeptidase A (CPA) (1) has been obtained from a site specific modification of tyrosine 248 with Co(III) (2). The method developed for CPA has been extended to other enzymes and hormones in order to devg op an improved method for incorporating stable radioisotopes t Co) into proteins. The substitution-inertness of Co(III) provides the necessary stability in these derivatives (3). 

A more selective modification, with concomitant loss of activity, has been achieved using diazotized [ °S]sulfanilic acid (130,194). The free thiol groups were protected by mercuric chloride before the tyrosine modification was performed. It was found that an incorporation of 1.5 radioactive sulfur atoms per subunit inhibited the activity. The labeled peptide was isolated and partially sequenced. By comparison with the dogfish LDH sequence the labeled tyrosine corresponds to residue 220. However, this tyrosine is nowhere near the active center of the subunit. It is most improbable that it can be associated with catalysis. 

Price and Radda (338) found that N-acetylimidazole could acetylate up to six tyrosine residues without loss of activity or alteration of Km for substrate however, reaction of about one tyrosine per subunit results in desensitization toward GTP, but the response to ADP is not abolished even by extensive 0-acetylation. Essentially the same results are observed upon nitration with tetranitromethane (TNM). Acetylation does not grossly alter the molecular weight, as measured by sedimentation velocity, or the conformation, as determined by ORD. The GTP site is not protected by NADH alone, but is partially protected (25-50%) by GTP and is at least 75% protected by inclusion of both GTP and NADH in the reaction mixture. Piszkiewicz et al. (339) confirmed these findings by modification with TNM. The reaction is biphasic with initial rapid formation of one residue of 3-nitrotyrosine per subunit. The primary site of reaction is tyrosine-406 in the linear sequence (340). Later (338) the same effect was obtained with chicken GDH with both enzymes there is no influence on activation by ADP. Further, the pH optima of the enzymes are not influenced by the degree of nitration or the inhibition by GTP or activation by ADP (338). 

The above studies emphasize the ability of diazonium-coupling reactions to modify proteins with extremely high efficiency, but one of the limitations of this method is the lack of selectivity that can be obtained when there are multiple tyrosines on the surface of a single protein. This has not been problematic for the viral capsids shown above, as only one tyrosine is accessible on each monomer, but many applications demand higher levels of selectivity than allowed by these coupling reactions. To address this need, and to increase the substrate scope for bioconjugation reactions in general, a versatile Mannich-type reaction has been developed for tyrosine modification, Fig. 10.3-5 [25]. In this reaction, aldehydes and anilines are mixed to form... 

Fig. 10.3-5 Tyrosine modification using a when proteins are treated alone with either three component Mannich-type reaction. component, (b) The reaction conversion is (a) Aldehydes and anilines condense to listed for a number of anilines and aliphatic form imines in situ, which react with tyrosine amines using a-chymotrypsinogen A as the residues through an electrophilic aromatic substrate and formaldehyde as the aldehyde substitution reaction. No reaction occurs component.

Fig. 10.3-6 Tyrosine modification using commercially available lysine-reactive probes, (a) The aliphatic amino group reacts chemoselectively with NHS esters, leaving the aniline amino group free to participate in the Mannich reaction. On addition of formaldehyde and a protein target, tyrosine residues are modified, (b) Modification of...

Fig. 10.3-8 Tyrosine modification using palladium jr-allyl chemistry, (a) Allylic acetates (shown), carbonates, and carbamates can be activated by palladium(O) in aqueous solution to yield electrophilic 7r-allyl complexes. These species alkylate tyrosine residues with high...

Zeolite membranes are amenable by surface modification with a variety of chemical functional groups using simple silane chemistry that may provide alternative surface chemistry pathways for enzyme immobilization. In this context, Shukla et al. [338] have recently used a chemically modified zeolite-clay composite membrane for the immobilization of porcine lipase using glutaraldehyde to provide a chemical linkage between the enzyme and the membrane. The effects of pH, temperature, and solvent on the performance of such biphasic zeolite-membrane reactors have been evaluated in the hydrolysis of olive oil to fatty acids. Similarly, Algieri et al. [339] have immobilized tyrosinase on FAU membranes for the enzymatic conversion of the 1-tyrosine to 1-DOPA as an effective drug for Parkinson s disease treatment. This approach combines the active role of zeolite membrane as enzyme support and inhibitor suppressor. Moreover,... 

Neurotoxins present in sea snake venoms are summarized. All sea snake venoms are extremely toxic, with low LD5Q values. Most sea snake neurotoxins consist of only 60-62 amino acid residues with 4 disulOde bonds, while some consist of 70 amino acids with 5 disulfide bonds. The origin of toxicity is due to the attachment of 2 neurotoxin molecules to 2 a subunits of an acetylcholine receptor that is composed of a2 6 subunits. The complete structure of several of the sea snake neurotoxins have been worked out. Through chemical modification studies the invariant tryptophan and tyrosine residues of post-synaptic neurotoxins were shown to be of a critical nature to the toxicity function of the molecule. Lysine and arginine are also believed to be important. Other marine vertebrate venoms are not well known. 

Originally, for preparation of such conjugates the hydroxyl groups of monomethoxy-PEG (mPEG) were activated with cyanuric chloride, and the resulting compound then coupled with proteins (10). This approach suffers from disadvantages, such as the toxicity of cyanuric chloride and its limited applicability for modification of proteins having essential cysteine or tyrosine residues, as manifested by their loss of activity. 

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). 

Acetic anhydride is the only monocarboxylic acid anhydride that is important in modification reactions. The acetylation of the amino groups of proteins can be made relatively specific if the reaction is done in saturated sodium acetate, since the o-acetyltyrosine derivative is unstable to an excess of acetate ions (Fraenkel-Conrat, 1959). The tyrosine derivative rapidly hydrolyzes in alkaline reaction conditions, even in the absence of added acetate buffer (Uraki et al., 1957 Smyth, 1967). Treatment with hydroxylamine also cleaves any o-acetyltyrosine modifications, forming acetylhydroxamate, which can be followed by its purple complex with Fe3+ at 540 nm... 

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