Site-specific modification characteristics

Extreme caution must be used in interpreting the experimental data obtained from studies of the site-specific modification of enzyme sites. For an enzyme whose structure has been determined by X-ray crystallography, such as bovine pancreatic ribonuclease, the results of affinity labeling by a reactive nucleoside can be compared with the crystal structures of various enzyme-ligand complexes (271). The general characteristics of the haloacetyl class of affinity labels have been summarized (281). 

Enzyme-substrate complexes have been studied by kinetic analysis, chemical modification, inhibition of enzymes by specific compounds that interact with active sites, detection of characteristic spectral absorption bands during reaction of enzymes with substrates, and X-ray crystallographic analysis of enzymes combined with compounds which are in similar structure to the natural substrates. The interaction between enzymes and substrates has been analyzed by the concepts of lock-and-key" and "induced fit". The former presumes that the substrate surface must fit the enzyme surface like a key in a lock, while the latter refined theory assumes that binding of the substrate induces ( informational changes in the enzyme to provide a better fit. 

As described earlier, the ability to site specifically replace one amino acid with another genetically encoded residue provides extraordinary access to analyze protein structure and function. An area where it is often applied is in the assessment of the role of phosphorylation of side chains. Typically, two classes of mutants are made those that prevent modification (nonphosphorylatable) and those that are constitutive (nonhydrolyzable) phosphorylated mimics. For the former, the phosphorylatable residues Ser and Thr are replaced with Ala, and Tyr with Phe (Fig. 7.2-3). These are reasonably successful in many cases, although they can be misleading because they lack the hydrogen-bonding and polarity characteristics of the authentic residues [19]. More difficult is the substitution of a phosphoamino acid with one of the 20 encoded residues. Phosphoserine/threonine is commonly replaced with Asp or Glu residues (Fig. 7.2-4). However, Asp and Glu are deficient in several respects. First,... 

Information regarding the seismic wave transmission at SRS in terms of frequencies of interest is uncertain due to a lack of recent in-situ property soil data from the K-Reactor site vicinity. Therefore, possible modifications of the surface free-field motion due to site response characteristics have not been determined. WSRC is calculating site-specific estimates of ground motion... 

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