Metalloenzymes, modified

In many biocatalytic systems, the metal plays an important role at the active site (more than 50% of all known enzymes need a metal ion to be active), and usually the reaction intermediates reside on the metal ion in the enzyme. Bioinorganic catalysis is defined as a branch of catalysis dealing with processes performed with the aid of metalloenzymes, modified enzymes, and synthetic metal-containing molecules resembling the active site of metalloproteins. 

Several model systems related to metalloenzymes such as carboxypeptidase and carbonic anhydrase have been reviewed. Breslow contributed a great deal to this field. He showed how to design precise geometries of bis- or trisimidazole derivatives as in natural enzymes. He was able to synthesize a modified cyclodextrin having both a catalytic metal ion moiety and a substrate binding cavity (26). Murakami prepared a novel macrocyclic bisimidazole compound which has also a substrate binding cavity and imidazole ligands for metal ion complexation. Yet the catalytic activities of these model systems are by no means enzymic. 

RNA hydrolysis, 45 285-287, 297-299 metalloenzymes, 45 251-252 bleomycin, 45 252-260, 299 nucleic acid hydrolysis metal ions and, 45 283-285 by oligonucleotide modified with metal complexes, 45 297-299 of phosphodiesters, 45 251, 287-297 by ribozymes, 45 285-287 cleavage by iron bleomycin, 43 140 polymerase, arsonomethyl phosphonate analogue, 44 201-202 substructures, 43 133-134 transfer... 

A large number of investigations of the mechanism of electron transfer reactions of macromolecule-metal complexes in biological systems has been reported. These investigations were concerned with not only natural metalloenzymes such as cytochromes, ferredoxin, blue coppers, oxygenase, peroxidase, catalase, hemoglobin, and ruberodoxin, but also modified metalloenzymes 47). 

N-substituted iron porphyrins form upon treatment of heme enzymes with many xenobiotics. The formation of these modified hemes is directly related to the mechanism of their enzymatic reactivity. N-alkyl porphyrins may be formed from organometallic iron porphyrin complexes, PFe-R (a-alkyl, o-aryl) or PFe = CR2 (carbene). They are also formed via a branching in the reaction path used in the epoxidation of alkenes. Biomimetic N-alkyl porphyrins are competent catalysts for the epoxidation of olefins, and it has been shown that iron N-alkylporphyrins can form highly oxidized species such as an iron(IV) ferryl, (N-R P)Fe v=0, and porphyrin ir-radicals at the iron(III) or iron(IV) level of metal oxidation. The N-alkylation reaction has been used as a low resolution probe of heme protein active site structure. Modified porphyrins may be used as synthetic catalysts and as models for nonheme and noniron metalloenzymes. 

Bhandari et al. (56) modified a hit structure derived from the primary screening of various libraries (>300,000 compounds) on the zinc metalloenzyme phosphomannose isomerase from the yeast Candida albicans (CaPMI) to find enzyme inhibitors as jxitential antifungal agents. During primary screening only a 1296-member SP dipeptide pool library (Lll, Fig. 9.16) showed activity on the enzyme. Its deconvolution and analytical characterization led to the discovery of a by-product, derived from incomplete coupling, that showed activity on the enzyme. This compound (9.21, Fig. 9.16) showed a weak inhibitory activity on CaPMI (ICso = 40 xM) and was selected for further chemical profiling. 

Desrosiers, R.R., Nguyen, Q.T., and Beliveau, R. (1999). The carboxyl methyltransferase modifying G proteins is a metalloenzyme. Biochem Biophys Res Commun 261 7. 

The copper metalloenzymes are involved in oxygen-using reactions. These enzymes include cytochrome c oxidase (respiratory chain), lysyl oxidase (collagen synthesis), and dopamine [3-hydroxylase (neurotransmitter synthesis). Lysyl oxidase is a small protein with a molecular weight of 32 kDa. This enzyme contains an unusual modification, namely cross-linking between two different parts of its polypeptide chain. The cross-linked region consists of a structure called lysine tyrosylquinone (Klinman, 1996). Two amino acids are involved in this cross-linked structure, and these are Lys 314 and Tyr 349. Lysine tyrosylquinone is used as a cofactor and is necessary for the catalytic activity of the enzyme. Other copper metalloenzymes contain a related cofactor, namely 2,4,5-tiihydrox5q5henylalanine (topaquinone, TPQ). Serum amino oxidase is a copper metalloenzyme that contains TPQ. TPQ consists of a modified residue of phenylalanine. The copper in the active site of the enzyme occurs immediately adjacent to the TPQ cofactor. 

A method for the preparation of ODNs containing terminal 2 -S,3 -0-cyclic phosphorothiolate has been described. Such modified ODNs would be of use to study metal interactions with metalloenzymes. 5 -Deoxy-5 -thioguanosine-monophosphorothioate has been prepared and incorporated into the 5 -termi-nus of RNA using T7 RNA polymerase. This can be dephosphorylated (alkaline phosphatase) to leave RNA with a free 5 -SH for further modification. 3 -Phosphoselenoates undergo facile selenium mediated autoligation of DNA or RNA strands with 5 -deoxy-5 -iodo-ODNs. The method also provides a means for incorporation of heavy atoms into ODNs suitable for X-ray crystallography. 

In their classic study they were able to modify specifically tyrosine 248 in the zinc metalloenzyme, carboxypeptidase A, to give the azotyrosine derivative, arsanilazotyrosine 248 carboxypeptidase A (AA-CPA-Zn), shown in Figure 3A. The native Zn is shown explicitly in order to differentiate it from externally incorporated Co as will be discussed. They found that at intermediate pH s, where the enzyme exhibits maximal activity, the azotyrosine is chelated to the intrinsically bound active site zinc. A distinct red color is associated with zinc chelation in contrast to the yellow and orange colors of the enzyme due to the presence of the free azophenol (low pH) and azophenolate (high pH), respectively (7). 

The RNA polymerase is a metalloenzyme and contains Zn2+. The first nucleotide is often an ATP or GTP and the triphosphate is maintained unless the 50 end of the transcript is posttranscriptionally modified, as is the case in eukaryotes (Chapter 25). The RNA polymerization reaction is chemically very similar to DNA synthesis in that the nascent RNA strand is synthesized in the 50 to 30 direction. In this reaction, the 30 hydroxyl group of the existing RNA chain undergoes a nucleophile attack on the a-phosphate of the incoming nucleotide which then releases PPi. 

As usual, most of the work on the contributions of electronic factors to electron transfer rates has dealt with the transfer of electrons between widely separated donors and acceptors. Most of the literature in the time period surveyed has dealt with elegant experimental studies of synthetic, modified metalloenzyme or of photosynthetic donor-acceptor systems. 

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