Proteins selenomethionine replacement

Table 12.1. The genetic code presented in this table is very nearly universal. There are isolated exceptions in the genome of mitochondria, which is described later in this chapter. Beyond that, the genetic code has been expanded to include codons for two unusual amino acids that occur in a modest number of proteins. These amino acids are selenomethionine, in which an atom of selenium replaces the sulfur atom of methionine, and pyrrolysine, a cychzed form of lysine. For details, see A. Ambrogelly, S. Palioura, and D. Soil, Nat Chem Biol 3 29-35 (2007). 

Isolation of alkaline phosphatase from Escherichia coli in which 85% of the proline residues were replaced by 3,4-dehydro-proline affected the heat lability and ultraviolet spectrum of the protein but the important criteria of catalytic function such as the and were unaltered (12). Massive replacement of methionine by selenomethionine in the 0-galactosidase of E. coli also failed to influence the catalytic activity. Canavanine facilely replaced arginine in the alkaline phosphatase of this bacterium at least 13 and perhaps 20 to 22 arginyl residues were substituted. This replacement by canavanine caused subunit accumulation since the altered subunits did not dimerize to yield the active enzyme (21). Nevertheless, these workers stated "There was also formed, however, a significant amount of enzymatically active protein in which most arginine residues had been replaced by canavanine." An earlier study in which either 7-azatryptophan or tryptazan replaced tryptophan resulted in active protein comparable to the native enzyme (14). 

Direct methods work if the molecules, and thus the unit cells and numbers of reflections, are relatively small. Isomorphous replacement works if the molecules are large enough that a heavy atom does not disturb their structures significantly. The most difficult structures for crystallographers are those that are too large for direct methods and too small to remain isomorphous despite the intrusion of a heavy atom. If a medium-size protein naturally contains a heavy atom, like iron or zinc, or if a selenomethionine derivative can be produced, the structure can often be solved by MAD phasing (Section IV.E). [NMR methods (see Chapter 10) are also of great power for small and medium-size molecules ]... 

The X REL1 crystal structure, which consists of residues 52-316 corresponding to the N-terminal domain, was resolved using the SeMet multiple-wavelength anomalous dispersion method (9). The selenomethionines used for this method should be replaced with methionines to obtain the original form of the protein for simulations (see Note 2). 

The constmction of synthetic selenocysteine-containing proteins or selenium-containing proteins attracts considerable interest at present, mainly for the reason that it can be used to solve the phase problem in X-ray crystallography. Selenomethionine incorporation has been used mostly uutil now for this purpose. There are also two reports ou uew synthetic selenocysteine-containing proteins. In one case, the active site serine of subtUisin has been converted into a selenocysteine residue by chemical means, with the result that the enzyme gains a predominant esterase instead of protease activity. In the second case, automated peptide synthesis was carried out to produce a peptide in which all seven-cysteine residues of the Neurospora crassa metallothioueiu (Cu) were replaced by selenocysteine. The replacement resulted iu au alteration of both the stoichiometry and the affinity of copper binding. ... 

An alternative procedure, called molecular replacement, uses information about known structures that are believed to be similar to that of the species being investigated. The known structure is used to estimate the electron density of the unknown structure, which is then refined and improved. Another method of dealing with the phase problem is to introduce atoms which absorb radiation in the region of the incident X-rays, leading to a process called anomalous scattering . For proteins, a popular method is to replace S by Se by using selenomethionine in place of methionine. For nucleic acids, iodouracil or iodocytosine can be used in place of thymine and cytosine respectively. 

Nonstandard amino acids can be either incorporated globally at multiple sites within a protein or inserted at specific locations (1, 15). Global misincorporation of nonstandard amino acids can produce protein polymers with altered physical properties that confer, for example, varied tensile strengths and elasticities (16). These unique biomaterials can be used in many medical applications, such as altering properties associated with cell adhesion. In other applications, routine replacement of methionine by selenomethionine aids in X-ray crystal structure determination. 

In all forms the selenium occurs in a position that usually has sulfur, a result that is not too surprising given their relative positions in the periodic table. In most cases, however, the reason for the replacement is unknown, although we speculate below about the function of selenocysteine in two of the enzymes. The role of selenium in those proteins that contain selenomethionine is particularly perplexing because the selenomethionine is not present in stoichiometric amounts at one particular position in the amino acid sequence rather it is distributed throughout several methionine sites . ... 

The replacement of sulfur-containing amino acids in proteins by their selenium analogs has received some attention. For example, a variant of Escherichia coli was shown to indiscriminately incorporate selenium into its proteins l. In fact, the /8-galactosidase isolated from this system, which had 70-75% of its methionine residues replaced by the selenium analog, exhibited a virtually unaltered catalytic activity. Similarly, the indiscriminate incorporation of selenomethionine in proteins of the rat as well as E. coli suggests that the enzymes methionyl-tRNA synthetase and amino acid polymerase can accept both methionine and selenomethionine and their corresponding tRNA derivatives. There is some evidence which suggests that a similar phenomenon occurs with cysteine and selenocysteine ... 

As a starting point for this exploration, we have examined the replacement of methionine (Met, 2) by selenomethionine (SeMet, 3) in proteins of repeating unit sequence 4. The substitution of 3 for 2 in native bacterial proteins was demonstrated many years... 

Ingested selenium is absorbed in the intestine, mainly in the duodenum. Thence, it is bound to a protein and transported in the blood to the tissues, where it is incorporated into tissue protein as selenocysteine and selenomethionine in the latter process, selenium replaces the sulfur in the amino acids cysteine and methionine. Excretion of selenium is largely by way of the kidneys, although small amounts are excreted in the feces and in sweat. 

MALDI-TOF-MS in order to select target ions for tandem MS. Finally, the sequences of the selected selenopeptides were derived from ESI-CID spectra. This combined approach using ICP-MS, MALDI-MS, and ESI-MS allowed for the first time the identification of the heat-shock protein HSP12 (Af, = 11,693 u) in which the only methionine residue was replaced by selenomethionine (Fig. 15.2) [26]. 

The specificity of human 5 -methylthioadenosine phosphorylase is rather strict if compared with that of the enzyme purified from E. coli The replacement of the sulfur atom of 5 -methylthioade-nosine by selenium and the replacement of the methyl group by an ethyl one are the only substrate modifications compatible with enzymic activity. The rate of breakdown of 5 -methylselenoadenosine equals that of 5 -methylthioadenosine (see Fig. 8). This finding agrees with the generally accepted view that the enzyme systems that normally utilize sulfur metabolites also convert their selenium analogues, i.e. the interchangeability of methionine and selenomethionine has been demonstrated in protein synthesisas well as that of S-adenosylmethionine and Se-adenosylselenomethionine in polyamine biosynthesis. 

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